Anti irf3

Cells were stimulated with Poly(I:C) (25 µg/ml), LPS (100 ng/ml) and R848 (1 µg/ml) as described and whole cell lysates were subjected to SDS-PAGE followed by immunoblot analysis with an IκBα (Cell Signaling Technology), an anti-phospho-IRF3 (Cell Signaling Technology), an anti-IRF3 (Santa Cruz) and anti-β-actin (Sigma) antibodies.

Thrombin (T4648), all chemicals were purchased from Sigma-Aldrich Corp. (St. Louis, MO), unless otherwise indicated. Transfection was done with Lipofectamine 2000 (Invitrogen). Ni-NTA beads were purchased from Clontech (635660). Anti-MAVS (Cell signaling, #3993), anti-IRF3 (Santa Cruz, sc-9082), anti-Flag (Sigma, F3165), anti-HA (Sigma, H9658), anti-GAPDH (Santa Cruz, sc-25778), anti-TBK1 (Santa Cruz, sc-52957; Cell signaling, #3504), anti-IKKε (Cell signaling, #2690S), anti-NEMO (Cell signaling, #2686S), anti-His (cw00c28), anti-IκBα (Cell signaling, #4814), anti-p-IRF3 (Epitomics, 2562–1), anti-p-TBK1 (Abcam, ab109272), anti-p-IκBα (Cell signaling, #2859), anti-p-IKKα/β (Cell signaling, #2078), anti-IKKα (Cell signaling, #2682), anti-IKKβ (Cell signaling, #2370), anti-NAP1 (Proteintech, 15042-1-AP), anti-TANK (Bioworld, BS2231), anti-SINTBAD (Cell signaling, #8605) antibodies were purchased as indicated. Antisera against viperion and p54/56 were generated by immunizing mice with the full length recombinant protein produced in E. coli, at Beijing Biotop Biotechnology, China.

Cells were washed twice with ice-cold phosphate-buffered saline (PBS; Wisent), harvested and lysed in 10mM Tris-HCl, 100mM NaCl, 0.5% Triton X-100, pH7.6 with EDTA-free Protease Inhibitor Cocktail (Roche). Cell lysates were clarified by centrifugation at 13,000 g for 20 min at 4°C and subjected to sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Western blot analysis was performed using mouse anti-PHF5A (Abnova), anti-IRF3 (Santa Cruz), anti-TRAF3 (Santa Cruz), anti-RIG-I (Alexis Biochemicals), anti-ACTIN (Chemicon International), anti-TBK1 (Imgenex and Santa Cruz), anti-IKBKE (Santa Cruz), anti-TUBULIN (ICN), anti-GAPDH (RDI) and rabbit anti-SNRNP200 (Sigma-Aldrich), anti-SF3A1 (Santa Cruz), anti-RELA (Santa Cruz), anti-NHP2L1 (Abcam), anti-DDX3X (Bethyl), anti-DDX60 (Abcam), TRIF (Cell Signaling), anti-ISG56 (Novus Biologicals), anti-MDA5 (Alexis Biochemicals), anti-MAVS (Alexis Biochemicals), anti-IKBKE (eBioscience), STAT1 (ABCAM), STAT1 tyr701 (ABCAM), IFNAR1 (Santa Cruz) and anti-IRF3-P-ser386 (Abcam). HRP-conjugated secondary antibodies were from Bio-Rad. The chemiluminescence reaction was performed using the Western Lighting Chemiluminescence Reagent Plus (PerkinElmer).

Protein lysates were extracted from cells, mixed with SDS loading buffer and boiled for 5 min before Western blot analysis. Protein expression was detected with anti-ISG15 (1:1000, #2743, Cell Signaling, Beverly, MA, USA), anti-IRF3 (1:1000, sc-376455, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-GAPDH (FL-335) (1:2000, sc-25778, Santa Cruz, Biotechnology, Dallas, TX, USA). The secondary antibody used on all blots was horseradish peroxidase-lined Donkey anti-rabbit IgG (1:2000, 406401, Biolegend, San Diego, CA, USA). All blots were developed with Clarity™ Western ECL Substrate (Bio-Rad, Hercules, CA, USA) before imaging with a Bio-Rad ChemiDoc™ MP.

ChIP assays were performed with SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology Japan, Tokyo, Japan) with anti-IRF1 (Santa Cruz Biotechnology), anti-IRF3(Santa Cruz Biotechnology), anti-IRF5 (Abcam), anti-IRF7 (Santa Cruz Biotechnology), anti-IRF8 (Santa Cruz Biotechnology), anti-IRF9 (Santa Cruz Biotechnology), normal goat IgG (Abcam) and normal rabbit IgG (Cell Signaling Technology) antibodies. ChIP signals were quantified by quantitative PCR analysis with a 7500 real-time PCR system (Applied Biosystems). Values obtained for immunoprecipitated samples (percent (%) input DNA) were normalized to values for respective normal IgG. The specific primer pairs for the Irf5 promoter region and P2rx4 promoter region, respectively, are described below.
Region 1: 5′-ATTTCTCAGGCCCTGTCTAAAGTG-3′ (forward),
5′-GGCACAGAGAGAGTTAGAGGAAGA-3′ (reverse)
Region 2: 5′-TATGGAGTCTTTCTGCACCCTGT-3′ (forward),
5′-TTCCAAGAACGAAGAGTCCCCTA-3′ (reverse)
ISRE-1: 5′-GCTGGCTCGTTTCAAGAATATT-3′ (forward),
5′-CGTACCCTGTAGCCGTCTATT-3′ (reverse)
ISRE-2: 5′-TCTACAGCCTGAAAGTCTATCATTG-3′ (forward),
5′-AAGGAATCTGAGAGGTACACACTG-3′ (reverse)
ISRE-3: 5′-GATAGGGAGAGGCTCGTTCA-3′ (forward),
5′-TAAAAGCTCGGGACCTGGAA-3′ (reverse)
ISRE-4: 5′-TACTGACCTGCCTCTTTTAAGGACA-3′ (forward),
5′-CGGAAAGAACTTTGAACCTTGAG-3′ (reverse)

For western blotting, cells were lysed with RIPA lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH8.0, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate) containing EDTA-free protease inhibitor cocktail (Roche). After centrifugation, clear supernatant was collected, added with protein sample buffer to final concentration of 50 mM Tris-HCl pH6.8, 2% SDS, 10% glycerol, 30 mM DTT, 0.002% bromophenol blue, boiled for 10 min and separated on SDS-PAGE gel. Proteins were semi-dry transferred onto PVDF membrane, blocked with 5% skim milk and probed with antibodies as indicated. For immunostaining, cells seeded on chamber-slides were fixed with 4% paraformaldehyde, 0.5% NP-40 permeabilized and blocked with 5% normal donkey serum (Jackson ImmunoResearch), followed by staining using the indicated antibodies. Images were captured using LSM880 confocal microscope (ZEISS). Mouse anti-FLAG-tag (Sigma-Aldrich), anti-beta-actin (Sigma-Aldrich), anti-GAPDH (Santa Cruz Biotechnology) and rabbit anti-HA-tag (Cell Signaling Technology), anti-IRF3 (Santa Cruz Biotechnology) antibodies were used.