Anti β actin antibody

Cells were fixed with 10% (w/v) trichloroacetic acid (TCA; Sigma-Aldrich) to prevent proteolysis and incubated on ice for 30 min. After removal of TCA by centrifugation at 9000 g for 1 min, cell pellets were lysed in PAGE sample buffer (2% SDS ([Sigma-Aldrich], 2.5% 2-mercaptoethanol [Sigma-Aldrich], 10% glycerol [Sigma-Aldrich], and 50 mM Tris-HCl, pH 6.8) and boiled at 98°C for 2 min; 10 μg total protein was loaded into each lane of a 12% polyacrylamide-SDS gel, separated by SDS-PAGE, and transferred onto polyvinylidene fluoride membrane (Merck Millipore). Membranes were washed in blocking buffer (5% dry skimmed milk powder [Sigma-Aldrich] in PBS) and incubated overnight at 4°C with anti-γH2AX (1:1000), anti-FLAG antibody (1:5000; Sigma-Aldrich, RRID:AB_259529), or anti-β-actin antibody (1:1000; GenScript, Piscataway, NJ, RRID:AB_914102). After washing in PBS-T (0.05% Tween 20 [Sigma-Aldrich] in PBS), membranes were incubated in PBS-T containing horseradish peroxidase-conjugated goat anti-mouse IgG antibody (1:5000; Thermo Fisher Scientific, Waltham, MA, RRID:AB_228307) for 1 hr at room temperature. Membranes were washed with PBS-T and developed using Clarity Western ECL (Bio-Rad, Hercules, CA).

Antibody against IRF7 was purchased from Cell Signaling Technology (CST) (Beverly, MA, USA). Mouse anti-Flag antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody and anti-β-actin antibody were purchased from GenScript (Piscataway, NJ, USA). Horseradish peroxidase (HRP)-conjugated labeled Goat anti-mouse IgG secondary antibody was purchased from Jackson Immuno Research (West Grove, PA, USA). miR-541-3p mimics and inhibitors were purchased from Qiagen (Valencia, CA, USA). Polyinosinic-polycytidylic acid (poly(I:C)) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine 2000 transfection reagent was purchased from Invitrogen (Carlsbad, CA, USA).