Sc 13594

The protein extraction reagent and the extraction method used for the extraction of total protein from the cells of each group were as described previously (2 (link)). The proteins were separated by 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes for immunoblot analysis. Subsequent to blocking with 5% skimmed milk powder for 2 h at room temperature, the membranes were incubated with monoclonal rabbit anti-human HIF-1α (ab51608; 1:500; Abcam, Cambridge, UK), mouse monoclonal anti-human MMP-2 (sc-13594; 1:500 Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), polyclonal rabbit anti-human MMP-14 (ab109745, 1:500; Abcam), polyclonal rabbit anti-human Bcl-2 (sc-492-G; 1:500; Santa Cruz Biotechnology, Inc.) and GAPDH (ab14247; 1:3,000; Abcam) antibodies at 4°C overnight. The membranes were then incubated with peroxidase-conjugated secondary antibodies (EMD Millipore, Billerica, MA, USA) for 1 h at room temperature. The specific protein bands on the membranes were visualised by the enhanced chemiluminescence method and analysed using the Tanon 3500 (Tanon 3500R) Gel Imaging System (Jinglai Laboratory Equipment Co., Ltd., Shanghai, China). The absorbance of each band was measured, and the ratio of each target band to GAPDH was used to show the expression of each target protein.

WER1-Rb-1 cells were harvested and lysed with RIPA buffer (Beyotime, Jiangsu, China). Protein concentrations were determined by a BCA protein assay kit (Beyotime) according to the manufacturer's instructions. Proteins were separated using 12% SDS-polyacrylamide gels and transferred from the gel to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk and then exposed to the following primary antibodies: MMP-2 (1 : 1000, sc-13594, Santa Cruz), MMP-9 (1 : 1000, sc-6841, Santa Cruz), and anti-GAPDH (1 : 1000, sc-365062, Santa Cruz). The blots were rinsed with 0.1% TBST and incubated with horseradish peroxidase-(HRP-) conjugated corresponding secondary antibody (1 : 5000) for 1 h at room temperature. GAPDH was used as a control, and all the antibodies were purchased from Santa Cruz Biotechnology.