Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.
Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2′-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.
In mammals, cell cycle progression is dependent upon highly ordered events controlled by cyclins and cyclin-dependent kinases (Cdks)1. Through its cytoplasmic, nuclear, and centrosomal localization, CyclinB1/Cdk1 is able to synchronize different events in mitosis such as nuclear envelope breakdown and centrosome separation2. CyclinB1/Cdk1 protects mitotic cells against apoptosis3 and promotes mitochondrial fission, a critical step for an equal distribution of mitochondria to the newly formed daughter cells4.
In proliferating mammalian cells, mitochondrial ATP is generated via oxidative phosphorylation (OXPHOS) machinery (electron transport chain), which is composed of 5 multi-subunit complexes; complex I – complex V (CI-CV). Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase or complex I (CI) is the largest and least understood of the five complexes5. The complex consists of 45 subunits, 14 of which form the catalytic core. Once assembled, the complex assumes an L-shaped structure with one arm protruding into the matrix and the other arm embedded in the inner membrane6,7. Mutations in CI subunits are the cause of a variety of mitochondrial disorders8. A functionally efficient CI in OXPHOS is required not only for overall mitochondrial respiration9, but also for successful cell cycle progression10. Unravelling the mechanisms underlying the functioning of this membrane-bound enzyme complex in health and disease could enable the development of novel diagnostic procedures and advanced therapeutic strategies. In a recent study, we have found that the CyclinB1/Cdk1 complex translocates into mitochondria in the (Gap 2) G2/(Mitosis) M phase and phosphorylates CI subunits to enhance mitochondrial energy production, potentially to offset increased energy needs of cells during cell cycle11. Here we showcase experimental procedures and strategies that can be used to study mitochondrial translocation of otherwise nuclear/cytoplasmic kinases, their mitochondrial substrates as well as functional consequences of their mitochondrial localization using CyclinB1/Cdk1 as an example.
The finding that the CyclinB1/Cdk1 complex translocates into mitochondria when needed prompted the studies of mitochondria-specific overexpression and knockdown of this complex. To achieve mitochondria-specific expression of proteins, one can add a mitochondria targeting sequence (MTS) in the N-terminus of the protein of interest. Mitochondria targeting sequences allow the sorting of the mitochondrial proteins into the mitochondria where they normally reside12. We have used an 87 base mitochondria targeting sequence derived from the precursor of human cytochrome c oxidase subunit 8A (COX8) and cloned it into Green Fluorescent Protein (GFP)-tagged CyclinB1 or Red Fluorescent Protein (RFP)-tagged Cdk1 containing plasmids in frame. This method allowed us to target CyclinB1 and Cdk1 into the mitochondria, specifically changing the mitochondrial expression of these proteins without affecting their nuclear pool. By fluorescently tagging these proteins, we were able to monitor their localization in real time. Similarly, we have introduced MTS into a plasmid containing RFP-tagged dominant negative Cdk1, which allowed us to specifically knock down the mitochondrial expression and functions of Cdk1. It is essential to distinguish between mitochondrial and nuclear functions of the kinases that have dual localizations like Cdk1. Engineering MTS into the N-terminal of these dual functional kinases offers a great strategy that is easy to be employed and effective.
Since Cdk1 is a cell cycle kinase, it is fundamental to determine the cell cycle progression when Cdk1 is localized into mitochondria. To achieve this, we have utilized a new method to monitor DNA content in cells. Traditional methods include using BrdU (bromodeoxyuridine), a synthetic thymidine analogue, which incorporates into the newly synthesized DNA during the S phase of the cell cycle to substitute thymidine. Then the cells that are actively replicating their DNA can be detected using anti-BrdU antibodies. One disadvantage of this method is that it requires denaturation of DNA to provide access for the BrdU antibody by harsh methods like acid or heat treatment, which may result in inconsistency among results13,14. Alternatively, we utilized a similar approach to monitor the actively dividing cells with a different thymidine analog, EdU. EdU detection does not require harsh DNA denaturation as mild detergent treatment enables the detection reagent to access the EdU in newly synthesized DNA. The EdU method has proven to be more reliable, consistent and with potential for high-throughput analysis15.
Finally, to determine the mitochondrial substrates of Cdk1, we used a proteomics tool called 2D-DIGE, which is an advanced version of classical two-dimensional gel electrophoresis. Two dimensional electrophoresis separates proteins according to their isoelectric point in the first dimension and molecular weight in the second. Since post-translational modifications such as phosphorylation affect the isoelectric point and molecular weight of the proteins, 2D gels can detect the differences between phosphorylation statuses of proteins within different samples. The size (area and intensity) of protein spots changes with the expression level of proteins, allowing quantitative comparison between multiple samples. Using this method, we were able to differentiate the phosphorylated proteins in wild type versus mutant mitochondria-targeted Cdk1 expressing cells. The specific protein spots that showed in the wild type but were missing in the mitochondria-targeted mutant Cdk1 preparation were isolated and identified via mass spectrometry.
In traditional 2D gels, triphenylmethane dyes are used to visualize the proteins on the gel. 2D-DIGE uses fluorescent protein labels with minimal effect on protein electrophoretic mobility. Different protein samples can be labeled with different fluorescent dyes, mixed together and separated by the identical gels, allowing the co-electrophoresis of multiple samples on a single gel16. This minimizes the gel-to-gel variations, which is a critical problem in gel-based proteomics studies.
1. Isolation of Mitochondria from Cultured Cells
2. Co-immunostaining of Cdk1, CyclinB1 and COXIV, a Mitochondrial Resident Protein
3. Sodium Carbonate Extraction of Intact Mitochondria
4. Separation of Inner and Outer Membranes of Mitochondria (Isolation of Mitoplasts)
5. Construction of Mitochondria-targeted GFP/RFP-tagged CyclinB1/Cdk1 Vectors and Confirmation of Their Mitochondrial Localization
6. Identification of Differentially Phosphorylated Proteins via 2D-DIGE
7. In Vitro Kinase Assay
8. Site-directed Mutagenesis to Generate Dominant Negative Cdk1 (D146N)
9. Determination of Cell Cycle Phase Lengths with EdU Incorporation Assay
Sub-mitochondrial localization of CyclinB1 and Cdk1
Sodium carbonate extraction is used to determine whether a protein is located inside the mitochondria or on the outside surface, namely outer membrane. Once a protein is shown to localize inside the mitochondria, further determination of sub-mitochondrial localization can be made via mitoplasting combined with protease digestion. To specify the sub-mitochondrial localization of CyclinB1 or Cdk1, mitoplasts were isolated by diluting mitochondria in hypotonic buffers with decreasing concentrations of the osmotic sucrose from 200 mM to 25 mM. The outer membrane begins to rupture at 150 mM of sucrose, while the inner membrane remains intact until the final concentration at 25 mM of sucrose (Figure 1A). In combination with mitoplasting, protease protection assay can be performed using trypsin to digest exposed proteins following outer membrane rupture. This will result in digestion of intermembrane space proteins. If the protein of interest is protected from trypsin digestion, this indicates mitochondrial matrix localization of the protein. In this representative figure, mitochondrial matrix protein Hsp60, and intermembrane space protein Timm13 were used as sub-mitochondrial localization markers. Similar with Hsp60 but unlike Timm13, CyclinB1 and Cdk1 were protected from trypsin digestion, indicating their mitochondrial matrix localization (Figure 1B).
Mitochondrial Expression of MTS- and GFP-tagged CyclinB1 and Cdk1 Proteins
MTS is cloned in frame at the N-terminus of CyclinB1 or Cdk1 genes, which has GFP or RFP tags at their C-terminus. The resultant recombinant protein is mitochondria-targeted GFP- or RFP-tagged CyclinB1 or Cdk1. The list of the constructs generated and used in this study is shown in the figure. Using these constructs, overexpression of CyclinB1 and/or Cdk1 in the mitochondria was achieved, shown here by western blotting of the isolated mitochondrial fractions (Figure 2).
Potential Mitochondrial Targets of CyclinB1/Cdk1 Determined by 2D-DIGE
Cdk1 belongs to the serine/threonine (S/T) kinase family catalyzing the transfer of a phosphate from ATP to proline (P)-oriented S or T residues. A point mutation that replaces an aspartate (D) residue with asparagine (N) at position 146 of Cdk1 (D146N) generates a dominant negative (dn) Cdk1 mutant19. To study the function of mitochondrial Cdk1, a mitochondria-targeted Cdk1-dn protein was generated by constructing a plasmid (pERFP-N1-MTS-Cdk1-dn) containing a 29 amino acid-long mitochondrial targeting sequence (MTS) derived from the subunit VIII of the human cytochrome C oxidase linked to RFP-tagged dn-Cdk1. pERFP-N1-MTS producing mitochondria-targeted ERFP protein was used as an empty vector control. Mitochondrial phosphoproteins in G2/M cells transfected with both constructs were profiled by 2D gel analysis with pH 4-10 gel strips. Compared with empty vector transfectants (Figure 3, upper panel), a group of mitochondrial phosphoproteins was apparently absent or decreased in the Cdk1-dn transfectants (Figure 3, lower panel). Mass spectrometry analysis of the spots detected determined the identity of the proteins phosphorylated by Cdk1 in the mitochondria.
Cell Cycle Progression and Determination of Phase Lengths with EdU Pulse-chase Assay
To investigate the progression of cell cycle when mitochondrial CyclinB1/Cdk1 levels are increased, a pulse-chase labeling experiment using a thymidine analogue, ethynyl deoxyuridine (EdU) was performed to label the population of cells undergoing DNA synthesis20. This method allows the visualization of cell cycle captured over a 22 hr window by tracking the EdU-positive population when cells progress through S and G2/M phases and accumulate in G1 phase. The results show that labeled S phase cells progressed through G2/M phase and appeared in G1 phase as fast as 4 h in cells expressing wild type mitochondrial CyclinB1/Cdk1, as compared to 6 h in cells transfected with a vector control or mutant CyclinB1/Cdk1 (Figure 4A), indicating that enhancement of mitochondrial CyclinB1/Cdk1 accelerates cell cycle progression.
Figure 1. Mitochondrial CyclinB1/Cdk1 Localizes in the Matrix. (A-B) Sub-mitochondrial localization of CyclinB1 and Cdk1 detected by mitoplasting and protease protection assay, figure has been modified from Wang et al., 201411. The total (T), pellet (P), and supernatant (S) fractions were subjected to western blotting analysis with indicated antibodies. TIMM13 (an inter-space protein), and HSP60 (a matrix protein). Please click here to view a larger version of this figure.
Figure 2. Expression of Mitochondrial Cdk1 Constructs. Western blotting of mitochondrial fractions isolated from cells transfected with mitochondria-targeted CyclinB1 and/or wild type or dominant negative mutant Cdk1 (plasmids are indicated on the bottom11. pEGFP-N1-MTS and the pERFP-N1-MTS vectors were empty vector controls for MTS-CyclinB1 and MTS-Cdk1 respectively). Please click here to view a larger version of this figure.
Figure 3. Potential Mitochondrial Substrates of Cdk1. Mitochondrial proteins extracted from G2/M-peaked cells transfected with mitochondria-targeted empty vector (pERFP-N1-MTS, upper panel) or mutant Cdk1 (pERFP-N1-MTS-Cdk1-dn, lower panel) were labeled with Cy5 (green), separated by 2-D gel and phosphorylated proteins were stained with phosphoprotein dye (red). This figure has been modified from Wang et al. 201411. Please click here to view a larger version of this figure.
Figure 4. Mitochondrial Cdk1 Enhances G2/M Transition and Overall Cycle Progression.
Cell cycle analysis with EdU pulse-chase labeling. Scatter plot histograms of EdU-labeled cells were drawn for DNA content (X-axis) and EdU (Y-axis). The lower figures in each panel show the mean fluorescence intensity of the EdU labeled nuclei. The time points were indicated in h after the EdU pulse11. For all time points, gates displaying the following populations were drawn: G0/G1, S, and G2/M. For 6, 8, and 10-hr time points, EdU- labeled G1*, S/G2*, and G2/M* populations are shown. This figure has been modified from Wang et al., 201411. Please click here to view a larger version of this figure.
Sucrose Concentrations Used | |||||
No trypsin | 25 mM | 50 mM | 100 mM | 150 mM | 200 mM |
+ trypsin | 25 mM | 50 mM | 100 mM | 150 mM | 200 mM |
Table 1. Hypotonic Sucrose Buffers Used for Step 4.2
Step 1 | 30 V | 12 hr | Step and Hold |
Step 2 | 300 V | 0.5 hr | Step and Hold |
Step 3 | 1,000 V | 0.5 hr | Gradient |
Step 4 | 5,000 V | 1.33 hr | Gradient |
Step 5 | 5,000 V | 20,000 V hr | Step and Hold |
Table 2. Isoelectric Protocol Used for Step 6.4.5
Like the proteins destined for other subcellular organelles, the mitochondrial targeted proteins possess targeting signals within their primary or secondary structure that direct them to the organelle with the assistance of elaborate protein translocating and folding machines21,22. Mitochondria targeting sequences (MTS) obtained from exclusively mitochondrial resident proteins such as COX8 can be added to N-terminus of any gene sequence to target specific proteins into the mitochondria11,23,24. Here, CyclinB1 and Cdk1 genes were cloned into COX8 MTS containing vectors and upon expression, the recombinant CyclinB1 and Cdk1 were localized into the mitochondria. The advantage of this approach is that it enables the modification of gene expression in a particular sub-cellular compartment, in this case mitochondria, without changing the overall gene expression. With this strategy, mitochondria-specific functions of a nuclear kinase, Cdk1 were determined. Similarly, by adding MTS to a dominant negative Cdk1, knock down of the mitochondria-specific functions of Cdk1 was achieved, which allowed the identification of mitochondria specific targets of Cdk1, and enabled the analysis of functional consequences of mitochondrial absence of Cdk1 function. Overexpression of Cdk1 without the MTS tag results in enhanced expression of Cdk1 in both mitochondria and nucleus, and therefore complicates the further investigations of the consequences of mitochondria-specific actions of Cdk1.
However, this approach may not be suitable for all gene products as we have experienced a few failed attempts at relocating some kinases into the mitochondria via the addition of MTS tag. Some cell lines may be quite resistant to transfection, and finding the optimal protocol may be time consuming. Even when the cells are healthy and the transfection is successful, working with fluorescent tagged proteins exhibits some problems such as aggregation, incorrect localization, non-functional fusions, and weak signals.
Major limitations of the isolation of mitochondria and mitoplasts include the low yield and possible contamination from other cellular or sub-mitochondrial compartments. It is suggested that adherent cells are not ruptured efficiently by conventional chemical or mechanical methods, therefore, making it difficult to obtain high quantities of mitochondria from cultured adherent cells. Here, we utilized adherent cultures of MCF10A cells. To yield about 30 – 50 µg of mitochondrial protein, a starting amount of 3 – 5 x 107 cells were utilized. The homogenization step is another critical point for the final yield of mitochondrial preparations. Depending on the cell lines used, the number of strokes and/or time of homogenization may vary. For MCF10A cells, we observed that 10 min of homogenization by glass/glass homogenizer is required, while mouse embryonic fibroblasts (MEF) required only 3 – 5 min of homogenization. Since excessive homogenization can cause damage to the mitochondrial membrane and trigger the release of mitochondrial components, the standard conditions for each cell line should be determined by experience. The use of a glass-glass homogenizer increases the yield of mitochondrial preparations compared to glass/Teflon homogenizers. The starting amount of cells as well as freeze/thawing of cells may alter the number of strokes necessary to break open the cells. Finally, protein denaturation and aggregation may occur due to localized heating of the sample during homogenization. It is, therefore, essential to pre-chill the tissue grinder and keep samples on ice during this procedure.
Another method presented in this article is the use of EdU labeling to monitor cell cycle in real time. EdU is a modified thymidine analogue which is fluorescently labeled with a bright, photostable Alexa Fluor dye. EdU is efficiently incorporated into newly synthesized DNA. This method is a better alternative to the use of traditional labeling of proliferating cells with the nucleoside analogue, bromodeoxyuridine (BrdU). BrdU is incorporated into DNA during active DNA synthesis. The quantification of BrdU-labeled DNA requires DNA denaturation by relatively harsh methods such as high heat or high acidity to expose the BrdU molecules for BrdU antibody binding. The harsh treatment for DNA denaturation may affect the sample quality and is time consuming. With EdU, detergent permeabilization is generally sufficient for the EdU detection reagent to gain access to the DNA. Without the need to use harsh chemicals or heat for DNA access, the EdU method is easier to use, more accurate and consistent. Apart from the advantages EdU provides, there has been some concerns regarding the use of EdU to study proliferation. EdU showed a slight anti-proliferative activity at treatments over 72 hr, which can be reduced to negligible levels when EdU pulse was kept at 1 hr25.
One modification employed with EdU labeling is the fixing time and method. The kit suggested a 15 min fixation with the provided fixing solution. However, an additional fixation with 70% ethanol was utilized in this experiment. There are two reasons for the use of ethanol: 1) to follow the cell cycle progression over time, a time-point experiment was performed, where cells were collected every 2 hr. The cells were kept in 70% ethanol at 4 oC until all of the experimental time points have been completed. Actually, cells can be kept in 70% ethanol for longer (several weeks to months) if need be. 2) The cells used for the experiment were stably transfected with GFP-tagged Cdk1 and/or RFP-tagged CyclinB1. To separate EdU and PI signals from GFP/RFP fluorescence, ethanol fixation was utilized to denature the GFP and RFP proteins, and hence quench their fluorescence before performing the EdU and PI staining. Denatured GFP/RFP proteins are essentially totally non-fluorescent, presumably because the chromophore is no longer protected from quenching26,27.
To identify mitochondrial targets of Cdk1, 2D-DIGE method, which is superior to traditional 2D gels in many ways was used. In 2D-DIGE, distinct fluorescent dyes, e.g., Cy 3, 5 and 2, are used to label samples, which allows running up to 3 samples in one gel, reducing the variability without the need to run replicates like in standard 2D gel. The fluorescent dyes used in 2D-DIGE have a very high sensitivity of 0.2 ng/spot compared to that of triphenylmethane dyes at 100 ng/spot, thus, which requires smaller amount of proteins run on the 2D-DIGE gels with high spot resolution and publication quality gel scans. An automated software is used to detect, quantify and define differentially expressed proteins. Because of high spot resolution, differential protein expression in samples can be compared accurately using the software-aided spot quantification; a difference as low as 10% can be detected via 2D-DIGE, enabling visualization of post-translational modifications easily. The use of software-aided in-gel analysis also enables faster acquisition of data. However, since equipment, such as fluorescent scanners, are required for image acquisition, use of this method involve additional costs. Other limitations of 2D-DIGE include the poor representation of hydrophobic proteins as well as proteins with extreme isoelectric points and large molecular weights28,29. Further validation of results obtained with 2D-DIGE using alternative techniques, such as immunocytochemistry or western blot is required to confirm novel findings.
The authors have nothing to disclose.
This work was supported by NIH grants CA133402, CA152313 and Department of Energy Office of Science DE-SC0001271. We thank the University of California Davis Flow Cytometry Shared Resource Laboratory with funding from the NCI P30 CA0933730, and NIH NCRR C06-RR12088, S10 RR12964 and S10 RR 026825 grants and with technical assistance from Ms. Bridget McLaughlin and Mr. Jonathan Van Dyke for their help with the flow cytometry experiments.
32P ATP | PerkinElmer | BLU002001MC | |
Anti-mouse secondary antibody | Invitrogen | A-11003 | Alexa-546 conjugated |
Anti-rabbit secondary antibody | Invitrogen | A11029 | Alexa-488 conjugated |
ATP | Research Organics | 1166A | For in vitro kinase assay |
Cdk1 antibody | Cell Signaling Technology | 9112 | |
Cdk1 kinase buffer | New England Biolabs | P6020S | |
Click-iT EdU Alexa Fluor 488 Imaging Kit | Life Technologies | C10337 | For cell cycle analysis with EdU labeling |
COX IV antibody | Cell Signaling Technology | 4844S | For mitochondrial immunostaining |
Cyclin B1 antibody | Santa Cruz Biotech | sc-752 | |
CyclinB1/Cdk1 enzyme complex | New England Biolabs | P6020S | Avoid freeze/thaw |
CyDye DIGE Fluor Labeling Kit | GE Healthcare Life Sciences | 25-8009-83 | |
DIGE Gel and DIGE Buffer Kit | GE Healthcare Life Sciences | 28-9480-26 AA | |
Dimethylformamide | Sigma Aldrich | 319937 | DMF |
Dithiothreitol | Bio-Rad | 161-0611 | DTT |
dNTP | EMD Millipore | 71004 | For site-directed mutagenesis |
Dpn I enzyme | Stratagene | 200519-53 | For site-directed mutagenesis |
Dry Strip cover fluid | GE Healthcare Life Sciences | 17-1335-01 | Used as mineral oil |
EDTA | J.T. Baker | 4040-03 | |
EGTA | Acros Organics | 409910250 | |
Eppendorf Vacufuge Concentrator | Fisher Scientific | 07-748-13 | Used as vacuum centrifuge concentrator |
Fluoromount G | Southern Biotech | 0100-01 | Anti-fade mounting solution |
Fortessa Flow Cytometer | BD Biosciences | 649908 | For cell cycle analysis with EdU labeling |
Histone H1 | Calbiochem | 382150 | For in vitro kinase assay |
QIAquick Gel Extraction Kit | Qiagen | 28704 | For purifying DNA fragments from agarose gels |
Immobiline DryStrip Gels | GE Healthcare Life Sciences | 18-1016-61 | IEF (isoelectric focusing) strips |
Immobilized Glutathione | Thermo Scientific | 15160 | Glutathione-agarose beads |
Iodoacetamide | Sigma Aldrich | I1149 | IAA |
IPGphor 3 Isoelectric Focusing Unit | GE Healthcare Life Sciences | 11-0033-64 | IPGphor strip holders |
Isopropyl-b-D-thio-galactopyranoside | RPI Corp | 156000-5.0 | IPTG |
Leupeptin | Sigma Aldrich | L9783 | For cell lysis buffer |
Lipofectamine 2000 | Life Technologies | 11668027 | Transfection reagent |
Lysine | Sigma Aldrich | L5501 | For CyDye labeling |
Lysozyme | EMD Chemicals | 5960 | |
Mitoctracker Red/Green | Invitrogen | M7512/M7514 | Mitochondrial fluorescent dyes |
MOPS | EMD Chemicals | 6310 | |
pEGFP-N1 | Clonetech | 6085-1 | GFP-expressing vector |
Pfu | Stratagene | 600-255-52 | |
pGEX-5X-1 | GE Healthcare Life Sciences | 28-9545-53 | GST-expressing vector |
Phenylmethylsulfonyl fluoride | Shelton Scientific | IB01090 | PMSF |
Phosphate buffered saline | Life Technologies | 14040 | PBS |
Spectra/Por 4 dialysis tubing | Spectrum Labs | 132700 | as porous membrane tubing for dialysis |
Pro-Q Diamond Phosphoprotein Gel Stain | Life Technologies | P-33300 | For staining phosphoproteins on 2D gels |
Proteinase inhibitor cocktail | Calbiochem | 539134 | For cell lysis buffer |
QuikChange site-directed mutagenesis kit | Stratagene | 200519-5 | |
QIAprep Spin Miniprep Kit | Qiagen | 27104 | MiniPrep Plasmid Isolation Kit |
RO-3306 | Alexis Biochemicals | 270-463-M001 | Cdk1 inhibitor |
Rotenone | MP Biomedicals | 150154 | Complex I inhibitor |
Sodium carbonate | Fisher Scientific | S93359 | |
Sodium chloride | EMD Chemicals | SX0420-5 | For cell lysis buffer |
Sodium orthovanadate | MP Biomedicals | 159664 | For cell lysis buffer |
Sodium pyrophosphate decahydrate | Alfa Aesar | 33385 | For cell lysis buffer |
Sodium β-glycerophosphate | Alfa Aesar | L03425 | For cell lysis buffer |
SpectraMax M2e | Molecular Devices | M2E | Microplate reader |
Sucrose | Fisher Scientific | 57-50-1 | |
Tissue Grinder pestle | Kimble Chase | 885301-0007 | For mitochondria isolation |
Tissue Grinder tube | Kimble Chase | 885303-0007 | For mitochondria isolation |
Trichloroacetic acid solution | Sigma Aldrich | T0699 | TCA |
Tris | MP Biomedicals | 103133 | |
Triton-x-100 | Teknova | T1105 | |
Trypsin | Calbiochem | 650211 | |
Typhoon Imager | GE Healthcare Life Sciences | 28-9558-09 | Laser gel scanner fro 2D-DIGE |
Ubiquinone | Sigma Aldrich | C7956 |