Ripa protein lysate

The protein precipitates were extracted using an RIPA protein lysate (cat no. 89900, Thermo Fisher) containing a protease inhibitor phenylmethylsulfonyl fluoride (cat no. 36978, Thermo Fisher, USA). Protein concentrations were determined using a BCA kit (ThermoFisher, USA). Then, the protein was subjected to SDS polyacrylamide electrophoresis and transferred onto a PVDF membrane (cat no. IPVH00010, Millipore). The PVDF membrane was blocked with 5% skimmed milk at room temperature. Then, the PVDF membranes were incubated overnight at 4 ^∘C with primary antibodies diluted with 5% skimmed milk. The used primary antibodies were mouse anti-PCNA monoclonal antibody (1:2000, cat no. ab29, Abcam, USA) and rabbit anti-p21 polyclonal antibody (1:1000, cat no. 10355-1-AP, Proteintech, Chian). Rabbit anti-vinculin monoclonal antibody (1:5000, cat no. ab129002, Abcam, USA) was used as the loading control. Then, the appropriate secondary antibody (CoWin Biosciences) was incubated with the PVDF membranes based on the genetic origin of the primary antibody. A gel imaging system was used to scan the protein bands using ECL reagents, and vinculin was used as an internal reference.

RIPA protein lysate (Thermo Fisher Scientific, USA) was used to lyse bladder tissues and HMC-1 cells. After centrifugation at 12000 rpm for 15 minutes at 4° C, the concentration of protein was measured by BCA kit (Beyotime, Shanghai, China). Protein was added to the polyacrylamide gel for electrophoresis, and then protein on the gel was transferred to the polyvinylidene fluoride membrane (PVDF). After blocking with 5% BSA for 1 hour, rabbit monoclonal BTK (1:1000; Abcam, UK) was added to the membrane. After incubating overnight at 4° C, Goat Anti-Rabbit IgG H&L was added to the membrane and incubated for 1 hour at room temperature. The ECL substrate kit was used for the colour development of the band. Gel imager (Gel Doc XR+; Bio-Rad, USA) was used for observation and photography. Image J software (Version 1.0; National Institutes of Health, US) analyzed the grey value of the band and calculated the relative expression level of the protein.