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).