Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

Summary

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

Abstract

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.^1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Introduction

"Centromeres" were classically defined as regions of suppressed meiotic recombination in genetics and later recognized as the primary constriction of mitotic chromosomes, which plays an essential role in accurate chromosome segregation during mitosis. "Kinetochores" were described as the multilayered structures that bind to microtubules at the surface of centromeres, as revealed by electron microscopy; "kinetochores" were later defined as the macromolecular complex that localizes at the centromere of mitotic chromosomes. Despite a dramatic divergence of centromeric DNA sequences among vertebrates, kinetochore structure and composition are highly conserved. A dynamic interaction between spindle microtubules and the kinetochore is required for faithful segregation of chromosomes during mitosis, and defects in centromere-kinetochore function lead to aneuploidy and thereby cancer.

The centromere in most eukaryotes has no defined DNA sequence, but is composed of large arrays (0.3-5 Mb) of repetitive alphoid DNA consisting of 171-bp α-satellite DNA. Except in budding yeast, centromere identity is achieved not by the DNA sequence but by the presence of a special nucleosome that contains the histone H3 variant CenH3 (CENtromere Protein A [CENP-A] in humans).⁵ CENP-A nucleosomes localize to the inner plate of mammalian kinetochores⁷ and bind to the 171-bp α-satellite DNA. Active centromeres require CENP-A-containing nucleosomes to direct the recruitment of a constitutive centromere-associated network (CCAN) and the kinetochore proteins, which together regulate the attachment of chromosomes to the mitotic spindle and subsequent cycle progression through the spindle checkpoint.

In light of the above evidence, CENP-A has been proposed to be the epigenetic mark of the centromere⁸; however, the process by which CENP-A is incorporated into centromeric DNA and the factors responsible for this incorporation have not yet been well characterized. A short centromere-targeting domain (CATD) resides in the histone fold region of CENP-A, and replacement of the corresponding region of H3 with the CATD is sufficient to direct H3 to the centromere.⁹ Several studies suggested functional roles for post-translational modification (PTM) of CENP-A^12-16; however, the molecular mechanisms of these PTMs of CENP-A in the recruitment to centromeres have not yet been elucidated. We previously reported that CUL4A-RBX1-COPS8 E3 ligase activity is required for CENP-A K124 ubiquitylation and localization of CENP-A to centromeres.¹⁷

The discovery and characterization of kinetochore proteins have led to new insight regarding chromosome segregation.¹⁸ More than 100 kinetochore components have been identified in vertebrate cells by various approaches.^19,20 An understanding of how kinetochores assemble and function also comes from the characterization of the cellular functions of each centromere-kinetochore proteins and protein-protein network within cells.¹⁹ Direct visualization and advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.^1-4 Thus, developing and reporting methods of IIF staining and a quantitative IIF assay to specifically analyze each centromere-kinetochore protein is extremely important. In IIF staining, one should proceed with the staining protocol to avoid loss of the protein of interest or the rest of the cell. However, fixation destroys antigenic sites occasionally, and different antibody-antigen combinations work poorly with one fixative, but very well with another,²¹ and choice of fixative depends largely on the protein(s) of interest. Therefore, different fixative methods are crucial in IIF staining of centromere-kinetochore proteins.

Here optimized methods of indirect immunofluorescent (IIF) staining and an assay to address localization of endogenous centromere-kinetochore proteins, including CENP-A and Flag-tagged exogenous CENP-A proteins, and quantitation of these proteins in human cells have been developed. These methods can be applied to the analysis of centromere-kinetochore proteins in other species.

Protocol

1. Cell Culture and Transfection

1. Put a cover glass (22 mm x 22 mm) in a 6-well polystyrene plate. Optionally coat a cover glass with Poly-L-Lysine, 0.1% w/v, in water (see List of Materials/Equipment) to keep mitotic cells on the cover glass following the steps below:
   Note: Optimal conditions must be determined for each cell line and application.
   1. Aseptically coat culture surface with Poly-L-Lysine, 0.1% w/v, in water (0.4 ml/well of a 6-well polystyrene plate). Rock gently to ensure even coating of the culture surface.
   2. After 5 min, remove solution by aspiration and thoroughly rinse surface with sterile tissue culture grade water.
   3. Dry at least 2 hr before introducing cells and medium.
2. Seed HeLa cells¹⁷ or HeLa Tet-Off cells^17,22 on a cover glass (22 mm x 22 mm) put in a 6-well polystyrene plate. Check that cell density is 5.4 x 10⁵ per well. Culture cells in high-glucose DMEM with 10% FBS and 1% penicillin-streptomycin.
   Note: For optimal results, empirically determine the cell density to use in seeding.
3. Incubate the cells at 37 °C in an atmosphere of 5% CO₂ for 18 hr.
   Note: In the case of HeLa Tet-Off cells, transcription of the exogenous gene is active in the absence of the inducer (i.e., tetracycline/doxycycline) therefore culture cells without tetracycline/doxycycline and transfect transiently with the pTRM4 overexpression vector (Table 2), whose transcription is regulated by the TRE promoter (see below).
4. Eighteen hours after seeding, transfect cells as follows:
   1. Make solution A by mixing 1.5 µl siRNA oligo (20 µM annealed stock; Table 1) and/or 2.0 µg plasmid (Table 2) in 50 µl reduced serum medium (see List of Materials/Equipment), and incubate at RT for 5 min.
      Note: In this analysis, CA-UTR siRNAs (a mixture of 5' and 3' UTR siRNA; Table 1) were co-transfected for use in Protocols 3 and 4 (see Discussion).
   2. Make solution B by mixing 0.75 µl transfection reagent I (see List of Materials/Equipment) in 50 µl reduced serum medium, and incubate at RT for 5 min.
      Note: An optional step is the addition of 1.0 µl transfection reagent II (see List of Materials/Equipment).
   3. Mix solutions A and B together, and incubate at RT for 15 min.
   4. Wash the cultured cells once with PBS, and then add 500 µl reduced serum medium to each well of the 6-well polystyrene plate. Add the mixture of solutions A and B (i.e., RNA and /or DNA-lipid complex) directly to each of the individual well.
      Note: Final concentration is 3.3 µg/ml (plasmid); 50 nM (siRNA).
   5. Incubate the cells at 37 °C in an atmosphere of 5% CO₂ for 4.5 hr. Change the medium to high-glucose DMEM with 10% FBS and 1% penicillin-streptomycin.
   6. Incubate the cells at 37 °C in an atmosphere of 5% CO₂ for 48-72 hr after transfection.
      Note: For optimal results, the incubation period for cell growth before fixation must be determined empirically, and protein depletion and/or expression must be confirmed by Western blot analysis (see Protocol 6).
   7. If mitotic cell analysis is of interest, add paclitaxel (10 nM) to cultured cells 24 hr before fixation, or add TN16 (0.5 µM) to the cultured cells 2.5 hr before fixation.

2. Cell Fixation and Immunofluorescent Staining to Detect Endogenous Centromere-kinetochore Proteins (Paraformaldehyde Fixation)

1. Preparation of fixative, buffers, and reagents.
   1. Freshly prepare 50 ml of 4% paraformaldehyde solution in PBS, pH 7.4.
      1. Add 40 ml of 1× PBS to a glass beaker on a stir plate in a ventilated hood. Heat while stirring to approximately 60 °C. Add 2 g of paraformaldehyde powder to the heated PBS.
      2. Raise slowly the pH by adding 1 ml of 1 N NaOH in total, because the powder will not immediately dissolve.
         Note: The solution clears after the addition of NaOH.
      3. Once paraformaldehyde has dissolved, cool and filter the solution.
      4. Adjust the pH with 1 N HCl to pH 7.4 and the volume of the solution with 1× PBS to 50 ml.
         Note: Aliquots of the solution can be frozen or stored at 2-8 °C for as long as 1 week. The solution should be ice-cold or stored at 4 °C until it is to be used.
   2. Prepare 50 ml of buffer KB1, which consists of 10 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.5% BSA; and 0.5% Triton X-100.
      Note: BSA should be freshly added.
      Note: The buffer KB series are based on buffer KB described in previous reports.^1,2,4
   3. Prepare 50 ml of buffer KB2, which consists of 10 mM Tris-HCl, pH 7.5; 150 mM NaCl; and 0.5% BSA.
      Note: BSA should be freshly added.
   4. Prepare 1 ml of buffer KB3 containing DAPI (50 ng/ml).
   5. Prepare 100 ml of mounting medium, which consists of 1 mg/ml p-phenylenediamine; 10% PBS, and 90% glycerol.
      1. Adjust the pH of 1× PBS to 9.8 with 1 N NaOH, dissolve p-phenylenediamine in the solution, and then add glycerol.
      2. Store 1 ml aliquots at -80 °C. Protect from light.
2. Remove the culture medium by aspiration at 48-72 hr post transfection (see Protocol 1) for cell fixation. Rinse cells once with PBS. Apply PBS to the side of the culture wells to avoid disturbing the surface of the cells.
   Note: The optimal time point for cell fixation must be determined empirically.
3. Fix the cells in 4% paraformaldehyde in PBS for 30 min at 4 °C. Rinse the cells twice with buffer KB2 to sufficiently remove residual 4% paraformaldehyde.
4. Permeabilize the samples in buffer KB1 for 30 min at RT. Rinse the cells once with buffer KB2, and add buffer KB2 for 5 min at RT to additional block sites of nonspecific binding.
   Note: Buffer KB1 also contributes significantly to blocking.
5. Remove a cover glass (22 mm x 22 mm) from a 6-well polystyrene plate using forceps. Using a hydrophobic barrier pen (see List of Materials/Equipment), draw a pale green square or circle to form a hydrophobic barrier around each cover glass sample. Do not touch or get too close to the cells with the hydrophobic barrier pen. Put the cover glass in new 6-well polystyrene plate.
6. Dilute a primary antibody to centromere or kinetochore protein (dilution ratio of 1:100 to 1:200; see also List of Materials/Equipment) and either an anti-CENP-B antibody (dilution ratio of 1:400) or an anti-centromere antibody (ACA) (dilution ratio of 1:2,000) as a centromere location marker in buffer KB2.
   Note: For optimal results, the final concentration of the primary antibody in this solution must be determined empirically.
7. Apply a sufficient volume (ca. 30 µl) of the diluted primary antibody to immerse the cell sample. Incubate the cell sample for 1 hr at 37 °C. Rinse the cells 3 times with buffer KB2.
8. Using buffer KB2, dilute a fluorophore-conjugated secondary antibody (dilution ratio of 1:100 to 1:200) directed against each primary antibody.
   Note: For optimal results, the final concentration of the secondary antibody in this solution must be determined empirically.
9. Apply a sufficient volume (a drop of ca. 30 µl on the cover glass) of the diluted secondary antibody to immerse the cell sample. Incubate the cell sample for 1 hr at 37 °C. Rinse the cells 5 times with buffer KB2 during a period of 30 min (five 6 min washes).
   Note: For optimal results (i.e., for minimal loss of cells), the optimal washing condition must be determined empirically.
10. Apply a sufficient volume of buffer KB3 containing DAPI (50 ng/ml) to immerse the cell sample. Incubate the cell sample for 5 min at RT. Rinse the cells 1-2 times with buffer KB2.
11. Mount the cover glass that contains the cell sample onto the micro slide.
    1. Place a drop of mounting medium in the center of the micro slide.
    2. Remove liquid from the cell sample and, using hands or forceps, position the sample in the center of the micro slide. Avoid air bubbles.
    3. Remove excess mounting medium with a paper towel.

3. Cell Fixation and Immunofluorescent Staining of C-terminal Flag-tagged CENP-A Proteins (Acetone Fixation)

1. Preparation
   1. Prepare ice-cold 75% acetone.
   2. Freshly prepare 50 ml of PBS (pH 7.4) containing 0.5% skim milk and 0.5% BSA.
   3. Freshly prepare 50 ml of PBS (pH 7.4) containing 0.1% skim milk and 0.1% BSA.
   4. Prepare 1 ml of PBS (pH 7.4) containing DAPI (50-100 ng/ml).
   5. Prepare 100 ml of mounting medium as described in 2.1.5.
2. Remove the culture medium by aspiration at 48-72 hr post transfection (see Protocol 1) for cell fixation. Rinse cells once with PBS. Apply PBS to the side of the culture well to avoid disturbing the surface of the cells.
   Note: The optimal time point for cell fixation must be determined empirically.
3. Fix the cells in ice-cold 75% acetone, and incubate the cells for 10 min at -20 °C. Dry cells on the cover glass in a fume hood for 30-60 min at RT.
   Note: For optimal results, the length of time needed for fixation and cell drying must be determined empirically.
4. Using the hydrophobic barrier pen (see List of Materials/Equipment), draw a pale green square or circle to form a hydrophobic barrier around each cover glass sample. Do not touch or get too close to the cells with the hydrophobic barrier pen.
5. Block nonspecific binding sites on the cells by adding PBS containing 0.5% skim milk and 0.5% BSA for 5 min at RT.
6. Using PBS containing 0.1% skim milk and 0.1% BSA, dilute an anti-Flag antibody (1:1,000 dilution ratio) and either an anti-CENP-B antibody (dilution ratio of 1:200) or ACA (1:2,000 dilution ratio) as a centromere location marker.
   Note: For optimal results, the final concentration of the primary antibody in this solution must be determined empirically.
7. Apply a sufficient volume (ca. 30 µl) of the diluted primary antibody to immerse the cell sample. Incubate the cell sample for 1 hr at 37 °C. Rinse the cells 5 times with the blocking buffer during a period of 30 min.
   Note: Cells can be rinsed with PBS. For optimal results (i.e., to avoid loss of cells), the optimal washing conditions must be determined empirically.
8. Dilute a fluorophore-conjugated secondary antibody (dilution ratio of 1:100 to 1:200) directed against the each primary antibody in PBS containing 0.1% skim milk and 0.1% BSA.
   Note: For optimal results, the final concentration of the secondary antibody in this solution must be determined empirically.
9. Apply a sufficient volume (ca. 30 µl) of the diluted secondary antibody to immerse the cell sample. Incubate the cell sample for 1 hr at 37 °C. Rinse the cells 2 times with PBS containing 0.1% skim milk and 0.1% BSA.
   Note: PBS alone can also be used to wash the cells.
10. Apply a sufficient volume of PBS containing DAPI (50-100 ng/ml) to immerse the cell sample. Incubate the cell sample for 5 min at RT. Rinse the cells 1-2 times with PBS.
11. Mount the cover glass containing the cell sample onto the micro slide as described in 2.11.

4. Cell Fixation and Immunofluorescent Staining of N-terminal Flag-tagged CENP-A Proteins (Methanol Fixation)

1. Preparation
   1. Prepare ice-cold methanol.
   2. Prepare TBS (pH 7.4) containing 4% goat serum.
   3. Prepare 1 ml of TBS (pH 7.4) containing DAPI (50-100 ng/ml).
   4. Prepare 100 ml of mounting medium as described in 2.1.5.
2. Remove the culture medium by aspiration at 48-72 hr post transfection (see Protocol 1) for cell fixation. Rinse cells once with TBS. Apply TBS to the side of the culture wells to avoid disturbing the surface of cells.
   Note: The optimal time point for cell fixation must be determined empirically.
3. Fix the cells in ice-cold methanol, and incubate the cells for 6 min at -20 °C. Rinse the cells twice with TBS to sufficiently remove residual methanol.
4. Using a hydrophobic barrier pen (see List of Materials/Equipment), draw a pale green square or circle to create a hydrophobic barrier around each cover glass sample. Do not touch or get too close to the cells with the hydrophobic barrier pen.
5. Block nonspecific binding sites on the cells by adding TBS containing 4% goat serum. Incubate for 10 min at RT.
6. Dilute an anti-Flag antibody (1:1,000 dilution) and either an anti-CENP-B antibody (dilution ratio of 1:200) or ACA (1:2,000 dilution ratio) as a centromere location marker in TBS containing 4% goat serum.
   Note: For optimal results, the final concentration of the primary antibody in this solution must be determined empirically.
7. Apply a sufficient volume (ca. 30 µl) of the diluted primary antibody to immerse the cell sample. Incubate the cell sample for 1 hr at 37 °C. Rinse the cells 5 times with the blocking buffer during a period of 30 min. For optimal results (i.e., the minimal loss of cells), the optimal washing condition must be determined empirically.
8. Dilute a fluorophore-conjugated secondary antibody (dilution ratio of 1:100 to 1:200) directed against each primary antibody in TBS containing 4% goat serum.
   Note: For optimal results, the final concentration of the secondary antibody in this solution must be determined empirically.
9. Apply a sufficient volume (ca. 30 µl) of the diluted secondary antibody to immerse the cell sample. Incubate the cell sample for 1 hr at 37 °C. Rinse the cells 3 times with the blocking buffer.
10. Apply a sufficient volume of TBS containing DAPI (50-100 ng/ml) to immerse the cell sample. Incubate the cell sample for 5 min at RT. Rinse the cells 1-2 times with TBS.
11. Mount the cover glass that contains the cell sample onto the micro slide as described in 2.11.

5. Immunofluorescence Image Observation, Acquisition, Quantitation, and Analysis

1. Observe the cell sample through a motorized fluorescence microscope equipped with an 63X and 100X oil immersion lens, an external compact light source, and a digital CCD camera.
2. Perform image acquisition and processing, including deconvolution, by using Software A, or Softwares B1 and B2 (see List of Materials/Equipment). Please see Supplemental Code Files (5.2.1) for all commands used in Software A. For all commands used in Softwares B1 and B2, please see (5.2.2) in Supplemental Code Files.
3. Use a previously described method^23-25 to quantify signals of centromere-kinetochore proteins (e.g., remaining signals of CENP-A at the centromere) with the following minor modifications:
   1. Select area of the centromere-kinetochore proteins and that of the background as follows:
      1. Mitotic cell: (centromere-kinetochore region) Select area overlapping with chromosomes stained with DAPI; (background region) and area outside chromosomes but inside the identical single cell (i.e., cytosolic region).
      2. Interphase cell: (centromere-kinetochore region) Select area overlapping with chromatin stained with DAPI; (background region) and area outside chromatin but inside the identical single cell (i.e., cytosolic region).
         Note: See also Supplemental Code Files (5.2.1.4.3) or (5.2.2.6.4) for area selection with Software A or Software B2, respectively.
   2. Quantify the percentage of remaining signals at the centromeres by using Software A or B. For this task, use the following formula:
      Remaining signals of centromere-kinetochore protein at the centromere-kinetochore
      
      where s is the signal brightness of the selected area, which is confirmed by ACA or CENP-B staining; b is the background signal brightness; r_sample is the reference ACA or CENP-B signals for siRNA(s)-treated cells; and r_ctrl is the reference ACA or CENP-B signals for Luc siRNA-transfected cells.
      Note: In this analysis, CENP-B signals were used as reference signals for CUL4A and RBX1 as described in previous reports.¹⁷
      1. Use an Excel file for this calculation after copying and pasting the raw data from the signal quantitation software described above. See also Supplemental Code Files: (5.2.1.4) and (5.2.2.6) for details. One example of calculation is shown in Table 3.
   3. Analyze at least 20 cells to eliminate variation in staining and image acquisition for each measurement level. Optionally use "averaged value" of remaining centromere-kinetochore signals for each analyzed cell to compare these values among different centromere-kinetochore proteins.

6. Western Blot Analysis of Total Protein

1.  Resuspend the cells in denaturing buffer A (20 mM Tris-HCl, pH 7.4; 50 mM NaCl; 0.5% Nonidet P-40; 0.5% deoxycholate; 0.5% SDS; 1 mM EDTA; and complete EDTA-free protease inhibitor cocktail),²⁶ subject the suspension to a sonication and freeze-thaw process, and measure protein concentrations as follows:
   1. For the sonication process, add 50 µl of buffer A to the cells collected from two wells of a 6-well polystyrene plate at 48-72 hr post transfection (see Protocol 1). Operate a sonicator equipped with disruptor horn and microtip (see List of Materials/Equipment) for total 15 sec intermittent-pulse duration (duty cycle 50%) per one sample.
   2. For freeze-thaw process, freeze cells with liquid nitrogen and thaw cells at RT.
   3. Measure protein concentrations using a commercial protein assay reagent I or II (see List of Materials/Equipment).
      Note: Lysates are diluted with ratio of 1:10 in the measurement of protein concentrations with either reagent I or II. At this dilution, the SDS present in buffer A shows little or no interference in this measurement.
2. Mix the lysate containing 20-30 µg of total protein with 2× or 4× SDS-PAGE loading buffer.²⁷ Boil the samples for 5 min and then load them on a 12.0%-15.0% denaturing SDS-polyacrylamide gel for electrophoresis.
3. Transfer the proteins separated by SDS-PAGE onto a PVDF membrane by using a Western blotting method described previously.^17,24,27-31
   1. Block the nonspecific binding sites on the membrane with 5% non-fat milk in 1× PBS, and then incubate the membrane with solutions of diluted primary antibodies for 1 hr at RT. See List of Materials/Equipment for detailed information (e.g., dilution ratio) of each primary antibody.
   2. After washing the membrane 3-4 times (each 3- to 5-min incubation with shaking) with PBS-T buffer (1× PBS and 0.1% Tween-20), incubate the membrane in a mixture of near-infrared (IR) fluorescent dye-conjugated secondary antibodies (dilution ratio of 1:20,000), DyLight-conjugated secondary antibodies (dilution ratio of 1:20,000), and/or horseradish peroxidase (HRP)-conjugated secondary antibodies (diluted in PBS-T; dilution ratio of 1:10,000) for 1 hr at RT. See List of Materials/Equipment for detailed information (e.g., dilution ratio) of each secondary antibody.
4. Wash the membrane 3 times, and then scan the membrane to analyze the proteins with the infrared imaging system and/or the chemiluminescence imager for immunoblot detection (see List of Materials/Equipment).
   1. For using the infrared imaging system, see (6.4.1) in Supplemental Code Files.
   2. For using the chemiluminescence imager, see (6.4.2) in Supplemental Code Files. Use an ultra-sensitive enhanced chemiluminescent (ECL) substrate (see List of Materials/Equipment) for this system.

Results

Immunofluorescence analysis of endogenous CENP-A supports the hypothesis that CUL4A-E3 ligase is required for localization of CENP-A to centromeres
Our recent studies showed that CUL4A-RBX1-COPS8 E3 ligase activity is required for ubiquitylation of lysine 124 (K124) on CENP-A and localization of CENP-A to centromeres.¹⁷ Initially, the interphase-centromere complex (ICEN) was isolated by anti-CENP-A:native chromatin immunoprecipitation,^32-34 and i...

Discussion

In recent years many studies have developed different quantitative microscopy assays for fixed cells.⁴² Progress in centromere-kinetochore biology often requires an understanding of the centromere-specific or kinetochore-specific function of proteins of which subcellular spatial-temporal regulation reflects the changing functions of these proteins during cell cycle. Therefore, here we developed methods of IIF staining and a quantitative IIF assay to specifically analyze the relative levels of endogenous and ex...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lipofectamin 2000	Life Technologies/Invitrogen	11668	transfection reagent I
Lipofectamin RNAiMAX	Life Technologies/Invitrogen	13778	transfection reagent II
Opti-MEM I	Life Technologies/Invitrogen	31985	Reduced serum media, warm in 37 °C water bath before use
High-glucose DMEM (Dulbecco’s modified Eagle’s medium)	Life Technologies/BioWhittaker	12-604	high-glucose DMEM, warm in 37 °C water bath before use
Fetal Bovine Serum, certified, heat inactivated, US origin	Life Technologies/Gibco	10082	FBS (fetal bovine serum)
Poly-L-Lysine SOLUTION	SIGMA-SLDRICH	P 8920	Poly-L-Lysine, 0.1% w/v, in water
UltraPure Distilled Water	Life Technologies/Invitrogen/Gibco	10977	Sterile tissue culture grade water
Micro Cover glass (22 mm x 22 mm)	Surgipath	105	Cover glass (22 mm x 22 mm)
6 Well Cell Culture Cluster	Fisher/Corning Incorporated	07-200-83	6-well polystyrene plate
Penicillin, Streptomycin; Liquid	Fisher/Gibco	15-140	Penicillin-streptomycin
PAP PEN	Binding Site	AD100.1	Hydrophobic barrier pen (for a water repellant barrier in immunofluorescent staining)
Paclitaxel (Taxol)	SIGMA-SLDRICH	T7402	Taxol for mitotic cell analysis
TN-16, microtubule inhibitor (TN16)	Enzo Life Sciences	BML-T120	TN16 for mitotic cell analysis
BSA (bovine serum albumin)	SIGMA-SLDRICH	A7906	Blocking reagent
Triton X-100	SIGMA-SLDRICH	T8787	Detergent for permeabilization
Paraformaldehyde	SIGMA-SLDRICH	P6148	Fixation reagant
DAPI	SIGMA-SLDRICH	D9542	For nuclear staining
p-phenylenediamine	SIGMA-SLDRICH	P6001	For mounting medium
VWR Micro Slides, Frosted	VWR International	48312-013	Micro slides
Anti-CENP-A antibody	Stressgen/Enzo Life Sciences	KAM-CC006	Mouse monoclonal antibody; dilution ratio of 1:100 (IIF), 1:5000 (WB)
Anti-CENP-B antibody	Novus Biologicals	H00001059-B01P	Mouse monoclonal antibody; dilution ratio of 1:200 (IIF, methanol/acetone fixation)-1:400 (IIF, paraformaldehyde fixation)
Anti-CENP-B antibody	abcam	ab25734	Rabbit polyclonal antibody; dilution ratio of 1:200 (IIF, methanol/acetone fixation)-1:400 (IIF, paraformaldehyde fixation)
Anti-centromere antibody (ACA)	Fitzgerald Industries International, Inc.	90C-CS1058	Human centromere antiserum; dilution ratio of 1:2000 (IIF)
Anti-CENP-H antibody	Bethyl Laboratories	BL1112 (A400-007A)	Rabbit polyclonal antibody; dilution ratio of 1:200 (IIF)
Anti-CENP-H antibody	BD	612142	Mouse monoclonal antibody; dilution ratio of 1:200 (IIF)
Anti-CENP-I antibody	N/A, Dr. Katusmi Kitagawa	N/A, Dr. Katusmi Kitagawa	Rabbit polyclonal antibody; dilution ratio of 1:1000 (IIF); Niikura et al., Oncogene, 4133-4146 (2006)
Anti-KNL1 antibody	Novus Biologicals	NBP1-89223	Rabbit polyclonal antibody; dilution ratio of 1:100 (IIF)
Anti-Hec1 antibody	Novus Biologicals / GeneTex	NB 100-338 / GTX70268	Mouse monoclonal antibody; dilution ratio of 1:100 (IIF)
Anti-Hec1 antibody	GeneTex	GTX110735	Rabbit polyclonal antibody; dilution ratio of 1:100 (IIF)
Anti-Ska1 antibody	abcam	ab46826	Rabbit polyclonal antibody; dilution ratio of 1:100 (IIF)
Anti-Flag antibody	SIGMA-ALDRICH	F3165	Mouse monoclonal antibody; dilution ratio of 1:1000 (IIF), 1:5000 (WB)
Anti-Flag antibody	SIGMA-ALDRICH	F7425	Rabbit polyclonal antibody; dilution ratio of 1:1000 (IIF), 1:5000 (WB)
Anti-CUL4A antibody	N/A, Dr. Pradip Raychaudhuri	N/A, Dr. Pradip Raychaudhuri	Rabbit polyclonal antibody; dilution ratio of 1:3000 (WB); Shiyanov et al., The Journal of biological chemistry, 35309-35312 (1999)
Anti-RBX1 antibody	Cell Signaling	4397	Rabbit polyclonal antibody; dilution ratio of 1:2000 (WB)
Anti-GAPDH antibody	Chemicon	MAB374	Mouse monoclonal antibody; dilution ratio of 1:5000 (WB)
Alexa Fluor 488 Goat Anti-Mouse IgG	Life Technologies/Invitrogen	A11001	fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody)
Alexa Fluor 594 Goat Anti-Mouse IgG	Life Technologies/Invitrogen	A11005	fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody)
Alexa Fluor 488 Goat Anti-Rabbit IgG	Life Technologies/Invitrogen	A11008	fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody)
Alexa Fluor 594 Goat Anti-Rabbit IgG	Life Technologies/Invitrogen	A11012	fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody)
Non fat powdered milk (approved substitution for carnation powdered milk)	Fisher Scientific	NC9255871 (Reorder No. 190915; Lot# 90629)	Skim milk
Leica DM IRE2 motorized fluorescence microscope	Leica		motorized fluorescence microscope
HCX PL APO 63x oil immersion lens	Leica	LEICA HCX PL APO NA 1.40 OIL PH 3 CS	63X oil immersion lens
HCX PL APO 100x oil immersion lens	Leica	LEICA HCX PL APO NA 1.40 OIL PHE	100X oil immersion lens
Leica EL6000 compact light source	Leica		External compact light source for fluorescent excitation
ORCA-R2 Digital CCD camera	Hamamatsu	C10600-10B	digital CCD camera
Openlab version 5.5.2 Scientific Imaging Software	Perkin Elmer/Improvision		For image observation, acquisition, quantification, and analysis
Velocity version 6.1.1 3D Image Analysis Software	Perkin Elmer/Improvision		For image observation, acquisition, quantification, and analysis
Complete EDTA-free protease inhibitor cocktail	Roche	11873580001/11836170001	Protease inhibitor cocktail tablets
PlusOne 2-D Quant Kit	Amersham Biosciences	80-6483-56	Commercial protein assay reagent I for measurement of protein concentration (compatible with 2% SDS)
Bio-Rad Protein Assay	Bio-Rad	500-0006	Commercial protein assay reagent II for measurement of protein concentration (compatible with 0.1% SDS)
Immobilon-FL	EMD Millipore	IPFL00010	PVDF membrane for transferring
IRDye 800CW Goat Anti-Mouse IgG	LI-COR Biosciences	926-32210	IR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:20000 (IIF)
IRDye 680 Goat Anti-Rabbit IgG	LI-COR Biosciences	926-32221	IR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:20000 (IIF)
Goat anti-Mouse IgG DyLight 549	Fisher Scientific	PI35507	DyLight-conjugated secondary antibodyIR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:20000 (IIF)
Goat anti-Rabbit DyLight 649	Fisher Scientific	PI35565	DyLight-conjugated secondary antibodyIR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:20000 (IIF)
Goat anti-mouse IgG-HRP	Santa Cruz	SC-2005	HRP-conjugated secondary antibodyDyLight-conjugated secondary antibodyIR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:10000 (IIF)
Goat anti-rabbit IgG-HRP	Santa Cruz	SC-2004	HRP-conjugated secondary antibodyDyLight-conjugated secondary antibodyIR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:10000 (IIF)
Openlab version 5.5.2. Scientific Imaging Software	Improvision/PerkinElmer		Software A
Volocity version 6.3 3D Image Analysis Software (Volocity Acquisition)	PerkinElmer		Software B1
Volocity version 6.3 3D Image Analysis Software (Volocity Quantification)	PerkinElmer		Software B2
Branson SONIFIER 450			Sonicator
Branson Ultrasonics sonicator Microtip Step, Solid, Threaded 9.5 mm	VWR Scientific Products Inc.	33995-325	Disruptor horn for sonication
Branson Ultrasonics sonicator Microtip Tapered 6.5 mm	VWR Scientific Products Inc.	33996-185	Microtip for sonication
Odyssey CLx Infrared imaging System	LI-COR Biosciences		Infrared imaging system for immunoblot detection
Image Studio Analysis Software Ver 4.0	LI-COR Biosciences		Software C
Molecular Imager Versadoc MP4000 System	Bio-Rad		Chemiluminescence imager for immunoblot detection
Quantity One 1-D analysis software	Bio-Rad		Software D
SuperSignal West Femto Maximum Sensitivity Substrate	Thermo	34095	Ultra-sensitive enhanced chemiluminescent (ECL) substrate