Western blotting analysis

Western blotting analysis:

Cells were washed with pre-cooled 1×PBS and collected in 100 µL lysis buffer containing 1% PMSF (Biyuntian, China) at 4°C for 15 mins. The protein concentration in the cell lysate was measured using the BCA concentration detection kit (Biyuntian, China). Equal amounts of total proteins were resolved by SDS-PAGE electrophoresis, and transferred to PVDF membranes using a Mini Trans-Blot cell (Bio-Rad, Hercules, CA) in transfer buffer [25 mM Tris base, 0.2 M glycine, and 15% (v/v) methanol] at 20 V, overnight at 4 °C with gentle stirring. The immunoblot was blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST), incubated with the primary antibody at 4°C overnight. After washing in TBST three times, blots were incubated with the corresponding HRP-conjugated secondary antibodies at room temperature for 1 h, followed by three washes with TBST. Antibodies for p-IRF3 (4947S), IRF3 (1190S), Bax (2772S) and GAPDH (5174S) were purchased from Cell Signaling Technology and antibodies for Bcl2 (ab32124) were obtained from Abcam company. Protein bands were detected by Immobilon Western Chemiluminescent HRP Substrate (Millipore Corporation, USA) and visualized using the Chemi DocTM Touch Imaging System (Bio-Rad).