Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.
The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon β and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.
The ubiquitously and constitutively expressed transcription factor Interferon (IFN) Regulatory Factor 3 (IRF3) is critical for the first line of defense against pathogens mainly through the induction of IFNβ, but also through the induction of the chemokine (C-C motif) ligand 5 (CCL5) and several antiviral proteins including IFN-induced protein with tetratricopeptide repeats IFIT1/2/31-3. IRF3 activation has been reported following infection with numerous viruses, or exposure to polyinosinic-polycytidylic acid (poly I:C) or lipopolysaccharide (LPS)4. Importantly, most studied viruses have evolved mechanisms to evade the IRF3-mediated response, and thereby escape the host innate immune defense5. Thus, monitoring IRF3 activation is of great importance to understand the molecular mechanisms of the innate antiviral host defense, but also to identify the strategy used by viruses to counteract this response.
Many published reports however provide only a limited analysis of IRF3 activation performed by the monitoring of IRF3-target gene induction (IFNB1 and IFIT1) and/or luciferase reporter gene assay coupled to low resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) analysis of IRF3. However, numerous biochemical studies, analysis of the behavior of various IRF3 mutants and elucidation of IRF3 crystal structure 6-11 have contributed to establish that IRF3 is subjected to a complex set of sequential post-translational modifications by phosphorylation at multiple sites. The set of phosphorylation involved in IRF3 activation appears to be dependent on the stimulus and most likely on the cell type. In uninfected cells, IRF3 coexists as non-phosphorylated and hypophosphorylated species containing phosphoresidues, including Thr135 and Ser173, in the 1-198 aa N-terminal region6,12-14. Accumulation of this hypophosphorylated form of IRF3 is induced by stress inducers, growth factors and DNA-damaging agents6. Phosphorylation of Ser/Thr residues at the C-terminal region of IRF3 containing the transactivation domain is triggered following activation by viruses, poly I:C or LPS in a cell-type dependent manner15-17. C-terminal phosphorylation of IRF3 involves no less than 7 distinct phosphoacceptor sites organized in two main clusters, Ser385/Ser386 and Ser396/Ser398/Ser402/Thr404/Ser405, that each contribute to IRF3 activation through dimerization, nuclear accumulation, association with the CREB-binding protein (CBP)/p300 coactivators, DNA binding to IFN sensitive response element (ISRE) consensus sequences and transactivation of target genes9,10,17-19. Phosphorylation of Thr390 is also thought to contribute to virus-induced IRF3 activation20. Mass spectrometry analyses of IRF3 have demonstrated that Ser386, Thr390, Ser396 and Ser402 residues are directly phosphorylated by the inhibitor of κB kinase ε (IKKε)/ TANK-binding kinase 1 (TBK1) kinases9,10. Phosphorylation at the C-terminal residues is also required for termination of IRF3 activation through polyubiquitination and proteasome-mediated degradation10. This process is also dependent on the phosphorylation at Ser339, which is necessary for the recruitment of the propyl isomerase Pin110,11. IRF3 species containing at least phospho-Ser339/386/396 residues are considered hyperphosphorylated forms. The exact sequence and function of each site remains a matter of discussion 10,21. It is now clear that activated IRF3 does not represent a homogeneous state, but that different activated species exhibiting distinct phosphorylation or dimerization characteristics exist 10,22.
To provide a complete understanding of IRF3 activation in response to specific pathogens, it is thus necessary to characterize which of the activated species are induced. Induction of IRF3 target genes, IFNB1 and IFIT1, has proven to provide a reliable read-out for IRF3 activation. However, monitoring expression of these genes does not distinguish between different activation states of IRF3. A comprehensive analysis of IRF3 activation states in a particular setting relies on the detailed characterization of its phosphorylation and dimerization status10. Unphosphorylated (form I), hypophosphorylated (form II) and hyperphosphorylated (forms III and IV) IRF3 forms6,18,23 can be successfully resolved by reduced mobility in high-resolution SDS-PAGE analysis. Monomeric and dimeric IRF3 species can be efficiently identified by native-PAGE analysis. These approaches are greatly improved when used in combination with phosphospecific antibodies directed against distinct IRF3 phosphoacceptor sites.
Standard protocols allow a poor resolution of proteins that does not permit efficient separation of distinct IRF3 phosphorylated forms. Here, we describe in detail a procedure to achieve the highest resolution to monitor the induction of distinct virus-activated IRF3 species using SDS-PAGE coupled to native-PAGE in combination with immunoblot using total and phosphospecific antibodies. In vivo discrimination between the different activated forms of IRF3 is performed based on their mobility shifts observed on SDS-PAGE. Additionally, IRF3 monomer and dimer can be distinguished by non-denaturing electrophoresis. The combination of these two complementary techniques with immunoblot proves to be an affordable and sensitive approach to acquire all the necessary information for a complete analysis of phosphorylation-mediated activation of IRF3.
NOTE: The protocol is described here using A549 cells infected with Sendai virus (SeV). However, the protocol for SDS-PAGE and native PAGE also works with all human and murine cell types tested so far, particularly myeloid cells stimulated with various IRF3-activating stimuli 9,15,19,24,25.
1. Infection of A549 Cells
2. Preparation of Whole Cell Extracts (WCE)
3. Resolution of WCE by High Resolution SDS-PAGE
4. Analysis of IRF3 Dimerization by Native-PAGE
NOTE: This method was originally described by the group of Dr. T. Fujita27.
5. Immunoblot Analysis of IRF3 Species
Figure 1. Sequence of Immunoblot Analyses. The schematic describes the individual SDS-PAGE and native-PAGE gels required to detect the various IRF3 phosphorylated and monomeric/dimeric forms. The specific order of antibodies applied to the membrane resulting from each gel in the immunoblot procedure is described. Note that the anti-actin antibodies are used first to ensure equal loading of the samples before applying any other specific antibodies. The alternate sequence, with anti-actin antibodies being applied after the anti-phospho-IRF3 antibodies, also works. Stripping is used between anti-actin and anti-SeV antibodies, or between anti-phospho-IRF3 and anti-IRF3 antibodies because of overlapping size of the signals. Please click here to view a larger version of this figure.
Primary antibodies | Dilution | Dilution buffer | Incubation | Secondary antibodies | Comments |
Anti-Actin | 1/10,000 | PBS-T-BSA | 15 min at RT | Anti-mouse | Use after SDS-PAGE Diluted antibodyies can be reused several times if stored at 4 °C in the presence of 0.02 % Sodium Azide. |
Anti-IRF3-P-Ser386 | 1/200 | PBS-T-BSA | O/N at 4 °C | Anti-rabbit | Use after Native-PAGE. It is not recommended to reuse the diluted antibody |
Anti-IRF3-P-Ser396 | 1/10,000 | PBS-T-BSA | O/N at 4 °C | Anti-rabbit | Use after SDS-PAGE. It is not recommended to reuse the diluted antibody Anti-IRF3-P-396 is also available from Cell Signaling. Optimal dilution was defined as 1/1,000 for this antibody, but may vary between lots. |
Anti-IRF3-P-Ser398 | 1/10,000 | PBS-T-BSA | O/N at 4 °C | Anti-rabbit | Use after SDS-PAGE. It is not recommended to reuse the diluted antibody |
Anti-IRF3 full length | 1/7,500 | PBS-T-milk | 3 hr at RT | Anti-rabbit | Use after SDS-PAGE. Anti-IRF3 full length antibody can be used after native-PAGE, but it is less sensitive to detect the monomer. Diluted antibodies can be reused several times if stored at 4 °C in the presence of 0.02 % Sodium Azide. |
Anti-IRF3-NES | 0.5 μg/ml | PBS-T-milk | 3 hr at RT | Anti-rabbit | Use after Native-PAGE. Diluted antibodyies can be reused several times if stored at 4 °C in the presence of 0.02 % Sodium Azide. |
Anti-SeV | 1/14,000 | PBS-T-BSA | 3 hr at RT | Anti-rabbit | Use after SDS-PAGE. Diluted antibodyies can be reused several times if stored at 4 °C in the presence of 0.02 % Sodium Azide. |
NOTE: Dilution and buffer used for HRP-coupled secondary antibodies need to be optimized as it varies from one company to the other. |
Table 1. Specifications of Antibodies used in the Immunoblotting Procedure.
Figure 2 shows a typical immunoblot image of IRF3 detected with IRF3 total antibodies and IRF3-phosphospecific antibodies against Ser396 and Ser398 after resolution of WCE by high-resolution SDS-PAGE. In unstimulated A549 cells, IRF3 is detected as two bands at 50 and 53 kDa on the SDS-PAGE corresponding to the non-phosphorylated (form I) and the hypophosphorylated (form II) species of IRF3. Exposure of A549 cells to SeV for 3 – 9 hr results in a time-dependent shift to slowly migrating hyperphosphorylated forms III and IV, which are well distinguished from the forms I and II. The hyperphosphorylated bands migrate at 55-57 kDa. The forms III and IV are also specifically immunodetected using the phosphospecific antibodies against Ser396 and Ser398. The immunodetection of actin serves as a control of equal loading between the lanes. A control of SeV infection is shown by the accumulation of the virus nucleocapsid protein (N), which migrates at 60 kDa.
In Figure 3, the profile of detection of IRF3 obtained from WCE resolved by native-PAGE followed by immunoblot using anti-IRF3-NES antibodies and phosphospecific antibodies against Ser386 is shown. In unstimulated A549 cells, IRF3 is detected as a single band corresponding to the monomeric form. Upon infection with SeV for 3 – 9 hr, the levels of IRF3 monomer decrease with a concomitant accumulation of a slowly migrating band that corresponds to the dimeric form of IRF3. The phosphospecific antibodies against Ser386 only detect IRF3 dimer species.
Figure 2. Detection of Distinct IRF3 Phosphorylated Species (form I-IV) Induced by SeV Infection in A549 Cells. A549 cells were left uninfected or infected with SeV for the indicated times. WCE were analysed by high-resolution SDS-PAGE followed by immunoblot (IB) using anti-IRF3-Ser396 (IRF-3-P-Ser396) and anti-IRF3-Ser398 (IRF-3-P-Ser398) phosphospecific antibodies and anti-IRF3 full length protein antibodies. The infection was monitored using anti-SeV antibodies (the nucleocapsid N protein is shown). Anti-actin antibodies were used as a loading control. Please click here to view a larger version of this figure.
Figure 3. Monitoring of Phosphorylated/dimerized IRF3 Induction by SeV in A549 Cells. A549 cells were left uninfected or infected with SeV for the indicated times. WCE were resolved by native-PAGE and revealed by immunoblot using anti-IRF3-Ser386 (IRF-3-P-Ser386) phosphospecific antibodies and anti-IRF3-NES antibodies. Please click here to view a larger version of this figure.
The protocol we describe here consists of a combination of high-resolution SDS-PAGE and native-PAGE coupled to the use of several phosphospecific antibodies to distinguish the monomeric/dimeric and phosphoforms I-IV of IRF3. Appropriate detection of these IRF3 species is essential to fully characterize IRF3 activation in a specific setting. For instance, LPS stimulation of activated macrophages leads to the formation of dimeric, Ser396/398 phosphorylated IRF3 that exhibits a hypophosphorylated (form II), but not hyperphosphorylated (form III and IV), pattern in SDS-PAGE15. Using the described protocol, this profile can be distinguished from virus-induced hyperphosphorylated forms that require additional Ser339 phosphorylation in addition to Ser386 and Ser396 phosphorylation10. Importantly, this also allows discriminating between Ser396 phosphorylated species that do not exhibit dimerization, but are transcriptionally active10. Importantly, it needs to be noted that the form I-IV pattern of IRF3 phosphoforms applied to human IRF3. Murine IRF3 does not exhibit a similar pattern and therefore activation of IRF3 is characterized only by the use of phosphospecific antibodies in immunoblot after SDS-PAGE and by native-PAGE.
The achievement of a high resolution separation of the distinct phosphorylated species of IRF3 by SDS-PAGE requires specific parameters. For appropriate resolution, it is very important to use gels that have a minimum length of 16 cm. Even with this length of separation gel, it is key to let the migration front reach the bottom of the gel to obtain an appropriate separation of the different IRF3 forms. Also, the stacking gel must extend to a minimum of 1 cm below the comb to obtain well-defined bands. This is important, as the hyperphosphorylated forms of IRF3 migrate very close to one another. Poorly focused bands will result in lack of distinction between phosphorylated forms impairing their respective quantification. Additionally, the concentration of ammonium persulfate and TEMED vary from one SDS-PAGE protocol to another. Variation in these concentrations from the ones indicated here was found to significantly impair the resolution in the separation of the IRF3 species.
Detection of IRF3 dimer formation through the native-PAGE was originally described by the group of Dr. T. Fujita. This protocol uses buffer containing DOC that allows the dissociation of the IRF3 dimer from CBP/p30027. It is highly recommended to proceed to native-PAGE immediately after cell lysis as storage of the WCE at -80 °C was found to result in a significant alteration of the IRF3 monomer/dimer ratio. Additionally, it is very important that the pH of the upper and lower chamber buffers is adjusted to pH 8.4 at RT, and not at 4 °C, after mixing all components to achieve proper separation of the monomer vs dimer. A separation gel with a minimum length of 8.5 cm allows appropriate separation of IRF3 monomer and dimer. The ideal volume of sample plus loading buffer is around 8 µl for a 5 mm well as the resolution is better when the volume is kept to a minimum. Thus, it is important to use the lowest volume of lysis buffer to obtain WCE with a high concentration. However, it should be taken into account that efficient protein extraction requires lysis in at least 5x the volume of the cell pellet. If the volume of the samples exceeds the limit indicated above, this would result in poor resolution of the monomer/dimer. This can be solved by adding a stacking gel containing 4% acrylamide, 62.5 mM Tris-HCl pH 6.8 (RT), 1% ammonium persulfate and 0.1% TEMED in ddH2O. It is very important to load the samples without disturbing banding. Loading slowly with a gel-loading tip close to the bottom of the well gives very good results.
Here, we describe the in vivo detection of phosphorylation at specific residues using currently available phosphospecific antibodies against Ser3869, Ser39619 and Ser39815. The use of IRF3 mutants and mass spectrometry analyses have confirmed that these residues are phosphorylated upon virus infection or overexpression of IKKε/TBK1 kinases and are critical for IRF3 activation6,9,10,19,21. The immunodetection of phospho-Ser396, phospho-Ser398 and total IRF3 after SDS-PAGE requires the use of two identical gels (Figure 1). Indeed, significant loss of sensitivity is observed when phosphospecific antibodies are used after the stripping of the membrane. Therefore, it is highly recommended to use phosphospecific antibodies first. Note that alternative primary and secondary antibodies may also work, but the specific dilutions and sequence of usage should be optimized. For IRF3 analysis, 30 µg of WCE is appropriate to obtain a significant detection of IRF3 phosphorylation by immunoblot using the indicated antibodies when using 4 mm width wells. Although in many cell types infected with various viruses 30 µg was found to be appropriate, this amount might need to be increased depending on cell type and stimulation. It is preferable to use fresh extracts for SDS-PAGE, but the detection of IRF3 phosphorylation using the phosphospecific antibodies described in this paper is stable enough to allow a round of storage of the WCE at -80 °C before analysis. The concentrations of phosphatase inhibitors described here were found to be sufficient when using A549 cells. Higher concentrations were not found to yield better results. However, these concentrations might need to be increased when studying IRF3 activation in cell types that exhibit higher phosphatase activity levels. Importantly, the phosphorylation of IRF3 and the underlying mechanisms are still far from being understood. Therefore, a full panel of Ser/Thr- and Tyr-phosphatase inhibitors is used to block possible yet uncharacterized activities that could affect the detection of IRF3 phosphorylation, dimerization and degradation. For detection of IRF3 monomer and dimer after most virus infections and in most cell types, 8 – 10 µg WCE was found to be sufficient. However, higher amount of proteins might be required for stimulation activating IRF3 less efficiently. The phosphospecific antibodies against Ser386 used in this study are recommended to be used in native-PAGE as the sensitivity in denaturing SDS-PAGE is very weak. Moreover, the phosphorylation at Ser386 is proposed to correlate with the dimeric form of IRF39, thus making the use of these antibodies in native-PAGE an appropriate strategy to confirm this concept in a specific setting. Of note, alternative antibodies are available from various companies and are claimed to efficiently detect phospho-Ser386 after SDS-PAGE. Phosphospecific antibodies against Ser396 have also been efficiently used to detect IRF3 phosphorylation after native-PAGE20. Importantly, Ser402 in the C-terminal cluster of phosphoacceptor sites was also found to be important for IRF3 activation and the phosphorylation of Ser402 was observed through mass spectrometry analysis of IKKε/TBK1-phosphorylated IRF310,21,28. However, despite two different attempts (NG, unpublished), no phosphospecific antibodies are currently available to follow the phosphorylation of this specific residue in vivo. Note that phosphospecific antibodies against Thr157 and Ser339 have also been described11,13. These additional phosphospecific antibodies could be used in a procedure of high-resolution SDS-PAGE similar to the one described here. In this case, additional large SDS-PAGE gels would be required for each antibody and processed the same way as gel #2 (Figure 1). To our knowledge no phosphospecific antibodies against Ser173 or Thr390 have yet been described, but they could be easily added to the protocol described here once they become available14,20.
IRF3 activity is terminated by proteasome-mediated degradation of hyperphosphorylated forms11,17. The protocol described here also permits the monitoring of IRF3 degradation when the kinetic of stimulation is prolonged and is coupled to the use of proteasome inhibitors (MG132, lactacystin or bortezomid). Typically, in A549 cells infected with SeV at 40 HAU/106 cells, IRF3 phosphorylation starts as soon as 2 hr post-SeV infection and IRF3 degradation starts after 12 hr. Proteasome-mediated degradation will be concluded from the observed diminution of forms III and IV levels at late time points that is reversed in conditions with proteasome inhibitors. This will also translate on native-PAGE in a diminution of IRF3 dimer levels that are recovered upon proteasome inhibitor treatment29. Importantly, the high-resolution technique presented here allows differentiating between the degradation process and a lack of activation. Indeed, both degradation and lack of activation of IRF3 result in the absence of detection of dimer/phospho-Ser386/396. However, lack of activation is associated with the detection of IRF3 monomer and of forms I and II, while these IRF3 species are not detected when IRF3 is degraded30.
Immunoprecipitation coupled with mass spectrometry-based analysis is a method of choice to detect the in vivo phosphorylation of IRF3 at distinct sites. This technique was used to confirm the phosphorylation of IRF3 at Ser173, Ser175, Ser386, Thr390, Ser396 and Ser402 residues10,20,21. However, mass spectrometry is not yet an affordable/benchside technique that can be easily used for daily analysis of IRF3 activation following several stimulations at different time points. Therefore, the procedure described here using SDS-PAGE coupled to native-PAGE in combination with immunoblot using total and phosphospecific antibodies constitutes a practical, affordable and sensitive approach to detect all currently defined forms of actived IRF3.
The authors have nothing to disclose.
The authors thank previous and current members of the laboratory for development of the protocols. The work was supported by funding from the Canadian Institutes of Health Research (CIHR) [grant # MOP-130527] and from the Natural Sciences and Engineering Research Council of Canada [NSERC-355306-2012]. NG is recipient of a Tier II Canada Research Chair. AR holds a studentship from the training program of the Respiratory Health Research Network from the Fonds de la recherche du Québec-Santé (FRQS).
F12/Ham | Life Technologies | 11765-054 | Warm in a 37°C bath before use. |
Fetal bovine serum | Life Technologies | 12483-020 | |
L-glutamine | Life Technologies | 25030-081 | |
D-PBS | Life Technologies | 14190-144 | For cell culture. |
Trypsin/EDTA 0.25 % | Life Technologies | 25200-072 | |
Sendai virus Cantell Strain | Charles River Laboratories | 600503 | |
Hepes | Bioshop | HEP001 | |
Sodium chloride (NaCl) | Bioshop | SOD001.5 | |
EDTA | Bioshop | EDT001 | |
Glycerol | Bioshop | GLY001.1 | Cut the extreminity of the tip and pipet slowly as it is very thick. |
IGEPAL CA-630 | Sigma-Aldrich | I7771 | Registred trademark corresponding to Octylphenoxy poly(ethyleneoxy)ethanol (Nonidet P-40) detergent |
Leupeptin | Bioshop | LEU001 | |
Aprotinin | Bioshop | APR600.25 | |
Sodium fluoride | Sigma-Aldrich | 201154 | |
Sodium orthovanadate | MP Biomedicals | 159664 | Activation of sodium orthovanadate 0.2M : 1) Ajust the pH to 10.0 using either 1 N NaOH or 1 N HCl. The starting pH of the sodium orthotovanadate solution may vary with lots of chemical. 2) The solution is yellow at pH 10.0. 3) Boil until colorless. 4) Cool to RT. 5) Reajust the pH to 10.0 and repat steps 3-4 until the solution remains colorless and stabilizes at 10.0. Store the activated sodium orthovanadate aliquots at -20°C. |
p-nitrophenyl phosphate disodium salt hexahydrate | Sigma-Aldrich | P1585 | |
Beta-Glycerophosphate | Sigma-Aldrich | G6376 | |
Bio-Rad Protein Assay Reagent | Bio-Rad | 500-0006 | Cytotoxic |
Acrylamide/Bis-Acrylamide (37.5 : 1) 40 % | Bioshop | ACR005 | Cytotoxic |
Tris-Base | Bioshop | DEO701 | |
Hydrochloric acid (HCl) | LabChem | LC15320-4 | Work under fume hood. Toxic and irritant. |
Sodium dodecyl sulfate (SDS) | Bioshop | SDS001.1 | Irritant. |
Amonium persulfate | Sigma-Aldrich | A3678 | |
TEMED | Invitrogen | 15524-010 | Toxic and irritant. |
Bromophenol blue | Fisher Scientific | B392-5 | |
Beta-mercaptoethanol | Sigma-Aldrich | M6250 | Work under fume hood. Toxic to the nervous system, mucous membranes. May be toxic to upper respiratory tract, eyes, central nervous system. |
Glycine | Bioshop | GLN001.5 | |
Sodium deoxycholate | Sigma-Aldrich | D6750 | |
Sodium hydroxide (NaOH) | Bioshop | SHY700 | Irritant. |
Nitrocellulose membrane (0.45mm) | Bio-Rad | 162-0115 | |
Acetic acid glacial | Bioshop | ACE222.4 | Work under fume hood. Toxic, irritant and flammable. |
Red ponceau | Sigma-Aldrich | P3504 | |
Potassium chloride (KCl) | Sigma-Aldrich | P3911 | For PBS composition for immunoblot. |
Na2HPO4 | Bioshop | SPD307.5 | For PBS composition for immunoblot. |
KH2PO4 | Sigma-Aldrich | P0662 | For PBS composition for immunoblot. |
Bovine serum albumin | Sigma-Aldrich | A7906 | For PBS-T-BSA composition for immunoblot. |
Non-fat dry milk | Carnation | ||
Poly sorbate 20 (Tween) | MP Biomedicals | 103168 | Cut the extreminity of the tip and pipet slowly as it is very thick. |
Anti-IRF-3-P-Ser386 | IBL-America | 18783 | Store aliquoted at -20oC. Avoid freeze/thaw. |
Anti-IRF-3-P-Ser396 | Home made19 | Store aliquoted at -80oC. Avoid freeze/thaw. | |
Phospho-IRF-3 (Ser396) (4D4G) | Cell Signaling Technology | 4947s | Store at -20oC. |
Anti-IRF-3-P-Ser398 | Home made15 | Store aliquoted at -80oC. Avoid freeze/thaw. | |
Anti-IRF-3-full length | Actif motif | 39033 | Store aliquoted at -80oC. Avoid freeze/thaw. |
Anti-IRF3-NES | IBL-America | 18781 | Store aliquoted at -20oC. |
Western Lightning Chemiluminescence Reagent Plus | Perkin-Elmer Life Sciences | NEL104001EA | |
LAS4000mini CCD camera apparatus | GE healthcare | ||
SDS-PAGE Molecular Weight Standards, Broad Range | Bio-Rad | 161-0317 | Store aliquoted at -20oC. |