β actin 60008 1 ig

T98G cells were washed with PBS and lysed with RIPA lysis buffer (Beyotime, Nantong, China) containing protease inhibitor (cOmplete Protease Inhibitor Cocktail Tablets; Roche). Equal amounts of protein were separated on 12 % SDS-PAGE and transferred onto an Immobilon-P PVDF membrane (Millipore, MA). The blots were blocked for 1 h with 2 % bovine serum albumin in Tris-buffered saline (20 mM Tris-HCl, pH 7.4, 0.15 M NaCl) containing 0.1 % Tween 20 (TBST) at room temperature (RT) and reacted overnight at 4 °C with the primary antibodies. The membranes were washed four times with TBST and then incubated with the secondary Ab for 1 h at RT. Horseradish peroxidase-conjugated goat anti-rabbit IgG (#31460) or goat anti-mouse IgG (#31430) secondary antibodies were used (Thermo Scientific Pierce, IL). The membranes were washed and visualized with BeyoECL Plus Chemiluminescent Substrate (Beyotime) for signal detection. Primary antibodies directed against p-IRF-3 (Ser396, 4D4G, #4947), p-TANK-binding kinase 1 (TBK1) (Ser172, D52C2, #5483), retinoic acid-inducible gene 1 (RIG-I) (D14G6, #3743), melanoma differentiation-associated protein 5 (MDA5) (D74E4, #5321), IkBα (#9242), and TBK1 (#3013) were obtained from Cell Signaling Technologies (Beverly, MA). IRF-3 (#11312-1-AP) and β-actin (#60008-1-Ig) antibodies were purchased from Proteintech Group, Inc. (Chicago, IL).