Sc 33641

At least 500,000 J-Lat 11.1 cells or 2 × 10⁶ CD4+ T cells were lysed per condition. Cells were washed with dPBS and lysed in ice-cold lysis buffer (100 mM NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA, 0.5% Nonidet P-40, protease and phosphatase inhibitor cocktail [Roche]). Cell lysates were denatured in Laemmli sample buffer and incubated for 5 min at 95 °C. The proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were blocked with 5% nonfat milk in Tris-buffered saline pH 7.4 and 0.5% Tween 20 (TBST) and then incubated with primary antibodies. After three washes with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Rockland Immunochemicals; 1:10,000) and detected using Western Lightning Plus-ECL reagent (Perkin Elmer). Signal intensity and densitometry analyses were conducted using ImageJ (NIH). The following antibodies were used: anti-Puromycin (12D10 Sigma-Aldrich; 1:1000), anti-GAPDH (ab8245, Abcam; 1:5000) and anti-DDX3 (ab128206, Abcam; 1:2000), anti-Phospho-IRF3 (Ser386) (37829, Cell Signalling Tech; 1:1000), anti-HSP70 (4872, Cell Signalling Tech; 1:1000), anti-IRF3 (sc33641, Santa Cruz; 1:1000), anti BIRC5 (sc17779, Santa Cruz; 1:1000) and anti-tubulin (B7, Santa Cruz; 1:3000).

The antibodies used and the sources are as follows: Antibodies against TBK1 (1:1,000, 3013), p-TBK1 (1:1,000, 5483), IKKε (1:1,000, 3416), p-IKKε (1:1,000, 8766), p-IRF3 (1:500, 4947) were purchased from Cell Signaling Technology. Antibodies against cGAS (1:2,000, 26416-1-AP), STING (1:2,000, 19851-1-AP), HA tag (1:2,000, 51064-1-AP), His tag (1:2,000, 66005-1-lg), and β-actin (1:20,000, 66009-1-lg) were purchased from Proteintech Group Inc. IRF3 antibody (1:1,000, sc-33641), RNF5 antibody (1:1,000, sc-81716), and c-Myc antibody (1,1,000, sc-40) were obtained from Santa Cruz Biotechnology. Monoclonal anti-Flag (1,1,000, F1804) antibody was purchased from Sigma-Aldrich, Inc. Anti-Calreticulin antibody (1,100, ab2907) was purchased from Abcam. Poly(dA:dT) (tlrl-patn) and cGAMP (tlrl-nacga 23–02) were purchased from Invivogen. Lipofectamine 2000 transfection reagent and Opti-MEM were from Thermos Fisher Scientific. Protease inhibitors were purchased from Roche. PMSF was purchased from Solarbio Life Sciences, Beijing, China.