Protein Immunoprecipitation and Western Blot Analysis

Cells were cultured in normoxia or hypoxia for at least 72 h and were lysed at 4 °C in NP-40 buffer containing protease inhibitor cocktail, 1 mM Na₃VO₄, 2 mM sodium fluoride and 1 mM phenylmethylsulfonyl fluoride. One to three milligrams of protein was immunoprecipitated at 4 °C overnight using either 30 μg of monoclonal anti-huCAIX antibody or 15 μg of monoclonal anti-MMP14 antibody covalently linked to CNBr-activated Sepharose 4B as described previously.^{66 (link)} In the case of co-IP with integrins, 1 mg of the cell lysate was immunoprecipitated at 4 °C overnight using 10 μg of monoclonal mouse anti- integrins β1and α2 and then incubated for 1 h at 4 °C with Protein A/G Agarose and UltraLink Resin (Cat no. 53132, ThermoFisher Scientific, Burlington, ON, Canada). The resin was extensively washed with NP-40 buffer pH 8.0 with 1% Tween-20, resuspended in sample buffer and boiled at 100 °C for 10 min under non-reducing conditions. The proteins in the supernatant were separated from the resin by centrifugation using a polypropylene spin column (Bio-Rad, Mississauga, Ontario, USA) for 1 min, at 4000 rpm. β-mercaptoethanol was added to the supernatant and eluates were boiled again. All samples were loaded on SDS–PAGE gels and western blots were performed as described above.