Electrophoretic transfer chamber

The A549 cells were collected 60 h post of infection. The cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer with protease inhibitors (Beyotime, Shanghai, China) added to collect total proteins. The proteins were loaded onto SDS-PAGE gels (10–15%), electrophoresed, and then transferred to polyvinylidene difluoride (PVDF) membranes (CWBiotech, Beijing, China) through an electrophoretic transfer chamber (Millipore, Temecula, CA, USA). The membranes were washed with Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) three times and blocked with 5% nonfat milk in TBST at 37 °C for 2 h. Subsequently, the membranes were incubated with primary antibodies at 4 °C overnight and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature (Goat anti-Rabbit IgG: Abcam, ab205718, 1:5000 dilution). The primary antibodies used were: GAPDH: CST, 5174, 1:1000 dilution; IRF1: CST, 8478, 1:1000 dilution. Finally, the immunoblot bands were visualized with an enhanced chemiluminescence (ECL) kit (Beyotime, China) and read using a chemiluminescence system (Thermo Scientific, Waltham, MA, USA).

Collected cells were lysed in Protein Extraction Reagent Type 4 (Sigma-Aldrich, Merck KGaA) with PhosStop phosphatase inhibitor and cOmplete protease inhibitor (Roche Diagnostics, Indianapolis, IN, USA). The protein concentration of cell lysates was measured with the Bradford reagent (Bio-Rad Laboratories, Inc.). Fifty microgram of total protein was electrophoresed through 10% SDS-PAGE gels and was transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc.) using an electrophoretic transfer chamber (Millipore, Temecula, CA, USA). The blots were incubated with primary antibodies overnight at 4°C followed by incubating with relevant HRP-conjugated secondary antibodies (Goat anti-mouse IgG antibody (Abcam ab6789) and goat anti-rabbit IgG antibody (Abcom ab97051)) for 1 hour at room temperature. Actin (sc-4778, Santa Cruz Biotechnology, Dallas, TX, USA) was used as a loading control. The signal was detected using an ECL western blotting detection kit (Promega, Fitchburg, WI, USA).