5 bromo 4 chloro 3 indolyl phosphate bcip nitro blue tetrazolium

Tumor tissues were homogenized in lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton and 1× complete protease inhibitor cocktail), and spined extract 10 mins at 14,000g in 4 ^oC centrifuge. Cells were lysed in lysis buffer, and sonicated briefly, centrifuged extract 15 mins at 14,000g in 4 ^oC centrifuge. Total protein samples (80 μg) were analyzed by 8% SDS-PAGE gel. The protein was ransferred to polyvinylidene difluoride (PVDF) membranes by a wet blotting procedure (100 V, 120 mins, 4^oC). After blocked with 5% blocking buffer, the membranes incubated with primary antibodies at 4^oC overnight using the following concentration: anti-GIT1 antibody (Epitomics, Burlingame, CA, USA) (1:1000), anti-Paxillin antibody (Epitomics, Burlingame, CA, USA) (1:1000) and GAPDH (1:3000), followed by alkaline phosphatase conjugated secondary antibody. Visualization of bound antibody was achieved with film (Kodak, Rochester, NY USA) by using 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) (Beyotime Institute of Biotechnology, Jiangsu, China) at several time points (2 mins, 10 mins and 2 hr). We chose the optimal conditions of GIT1/GAPDH signal image respectively and presented here. GAPDH acted as the internal control for normalization of gene expression data.