Irf3 d83b9

Samples were homogenized in lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 2 mmol/L EDTA, 2 mmol/L EGTA, 0.2% Triton X-100, 0.3% NP-40, 0.1 mmol/L PMSF, 25 mmol/L NaF). Proteins were separated by 10% SDS-PAGE under reducing conditions, then blotted onto nitrocellulose membranes. Membrane blockade was accomplished with 5% defatted milk in TBS-T (0.05 mol/L Tris, 0.15 mol/L NaCl, 0.05% Tween 20, pH 7.8). Thereafter, membranes were overnight probed at 4°C with primary antibodies in the same blocking solution or 5% BSA in TBS-T and then incubated with secondary HRP-conjugated antibodies for 1 h at room temperature. The following primary antibodies were used to detect specific proteins of interest: rabbit polyclonal anti-p-STAT1 (Tyr701) (Invitrogen, 44-376G), p-TYK2 (pTyr1054) (Origene, TA333304), and p-IKKε (Ser172) (Sigma Aldrich, 06-1340); rabbit monoclonal anti-p-TBK1/NAK (S172) (D52C2) XP^® (Cell Signaling Technology, 1,675,483), TBK1/NAK (E8I3G) (Cell Signaling Technology, 38,066), IKKε (D61F9) XP^® (Cell Signalling Technology, 3416), pIRF3 (Ser396) (4D4G) (Cell Signalling Technology, 4,947) and IRF-3 (D83B9) (Cell Signalling Technology, 4,302); monoclonal mouse anti-pIKBα (Santa Cruz, sc-8404). Anti-α-Tubulin (Sigma-Aldrich, MAB374) and anti-GAPDH (Millipore, MAB374) were used to assess protein loading homogeneity.

For western blot, anti-FAM26F C-terminal (ab194946; Abcam, Cambridge, MA, USA) rabbit polyclonal antibody (1:1000 dilution), IRF3 (D83B9; Cell signaling, Danvers, MA, USA) rabbit monoclonal antibody (1:1000 dilution), HBcAg (Santa Cruz Biotechnology, Dallas, TX, USA), GAPDH (FL-335; Santa Cruz Biotechnology, Dallas, TX, USA) rabbit polyclonal antibody (1:1000 dilution), and mouse-anti rabbit IgG HRP-conjugated (Sc-2357; Santa Cruz Biotechnology, Dallas, TX, USA)(1:10000 dilution) were used. The images were quantified by ImageJ software.