Mouse anti b raf f7

Total proteins were extracted from cell lysate with RIPA buffer (Thermo) and then quantified with a BCA protein assay kit (Beyotime), according to the specifications. Nuclear and cytoplasmic proteins were loaded into 10% sodium dodecyl sulfate (SDS) polyacrylamide gel, separately, and then transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore Corporation, Shanghai, China). After blocking with 5% non-fat milk, following primary antibodies were used to blot the special proteins at 4°C overnight: rabbit anti-IKKα/β pS180/S181 (1: 10000, Abcam), rabbit anti-NF-κB p65 (1: 5000, Abcam), rabbit NF-κB p65 pS536 (1: 10000, Abcam), rabbit anti-IκBα pS32 (1: 10000, Abcam), rabbit anti-NF-κB p100/p52 pS866 (1: 2000, Abcam), mouse anti-BRAF (F-7) (1: 200, Santa Cruz Biotechnology, Inc. Shanghai, China), rabbit anti-RET (1: 1000, Abcam), mouse anti-β-actin (1: 1,000, Proteintech Group, Inc. Wuhan, China), mouse anti-α-tublin (1: 10000, Proteintech). Goat anti-mouse and goat anti-rabbit secondary antibodies (1: 3000, Jackson Immuno Research Laboratories, Inc. West Grove, PA, USA) were used to incubate the proteins at 25°C for 1 h. An electrochemiluminescence (ECL, Millipore) system was used to expose the proteins.