Ab51745

Western blot was performed in cultured cells following various treatments. The protein lysates (1% NP40, 50 mM Tris, 5 mM EDTA, 1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 1 mM PMSF, 10 mg/ml aprotinin, 1 mg/ml leupeptin, and pH = 7.5) were measured by Bradford assay and the same amount of proteins was resolved on SDS-PAGE followed by an electric transfer to a PVDF membrane. The blots were blocked by 5% non-fat milk and incubated with primary antibodies, including HIF-1α (ab51608, 1:1000; Abcam, Cambridge, MA, United States), VEGF (ab51745, 1:2000; Abcam, United States) and GAPDH (ab9484, 1:1000; Abcam, United States). The blots were then incubated by appropriate HRP conjugated secondary antibodies, and signals were visualized by an enhanced ECL-based imaging system.

Cells were washed three times using cold PBS and lysed in RIPA buffer with protease inhibitors. Approximate 0.03 mg of protein was separated with 10% SDS-PAGE gel and blotted into nitrocellulose membranes. Then membranes were blocked with 5% nonfat dried milk blocking buffer at room temperature for 1 h and incubated with diluted primary antibodies (1:1000) against GAPDH (#5174, Cell Signaling Technology, 1:1000), E-cadherin (ab15148, Abcam, 1:1000), α-catenin (ab51032, Abcam, 1:1000), Vimentin (#5741, Cell Signaling Technology, 1:1000), Fibronectin (ab2413, Abcam, 1:1000), VEGFA (ab51745, Abcam, 1:1000) and ANGPT2 (ab65835, Abcam, 1:1000) at 4 °C overnight. Then membranes were washed by TBST 3 times and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000) at room temperature for 1 h. Protein bands were visualized by a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, USA) α-catenin.