Western Blot
Denatured protein samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime). Proteins were then transferred onto 0.2 μm PVDF membranes (Merck KGaA) and blocked with 5% skim milk for at least 1 h at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies. Anti-p-BTK was from Cell Signaling Technology, anti-BTK were from ABclonal, ant-p-I𝛋B𝛂, anti-I𝛋B𝛂, anti-p-p65, anti-p65, anti-GAPDH were from AiFang biological, which were diluted in TBST with 5% BSA at 1:1000. Diluted HRP-conjugated secondary antibodies (anti-rabbit/anti-mouse IgG, 1:10000, Beyotime catalog A0208/catalog A0216) were added to appropriate immunoblots and incubated at room temperature for 1 hour. Finally, immunoblots were visualized by ECL reagents (Beyotime). Immunoblot images were quantified and analyzed using ImageJ (v1.8.0).