Vegfa

Total protein was extracted using RIPA buffer containing proteinase inhibitor (Best Biological, Jiangsu, China). Western blot was conducted as previously described [12 (link)]. The primary antibodies for P-ERK1, ERK1, NF-κB1, RELA, P-mTOR, mTOR, VEGFA, PD-L1, P-P13K, P13K, P-AKT, AKT, TNF-α, P-EGFR, EGFR and HIF-1α were purchased from Bioss (Beijing, China). Total protein level was normalized to β-actin.

A total protein extraction kit (Solarbio) was used to extract bladder tissues or cells proteins. The protein samples were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (EMD Millipore). The membranes were incubated with primary antibodies (bFGF (1:1000, Bioss), HGF (1:1000, Bioss), VEGF-A (1:1000, Bioss), AKT (1:2000, Proteintech), pAKT (1:2000, Proteintech), GSK-3β(1:1000, Proteintech), pGSK-3β(1:2000, Proteintech), and GAPDH (1:2000, Proteintech)) overnight at 4 °C after blocked with 5% non-fat milk, then followed by a secondary horseradish peroxidase-conjugated antibody (anti-rabbit or anti-mouse (1:5000, Cell Signaling)) for 2 h at room temperature. Enhanced chemiluminescence system (ECL kit) was used to detect the immunoblot signals, which were quantified by scanning densitometry using the ImageJ analysis system (NIH).