Zymogram plus gelatin protein gels

Detection of MMP activity was done by gelatin zymography method. Cell culture supernatants were centrifuged for removing debris and 10 times concentrated (Microcon‐10 kDa, Millipore). Mouse sera were diluted 10‐20 times. Equal amounts of protein samples were run on 10% Zymogram Plus Gelatin Protein Gels (ZY00100BOX; Thermo Fisher Scientific) with 25 mM Tris buffer pH 8.3 containing 250 mM glycine and 0.1% SDS. After electrophoresis, gels were rinsed thrice for 30 minutes with washing buffer containing 2.5% Triton X‐100 and 30 minutes with H₂O, then incubated overnight at 37°C in 50 mM Tris‐HCl pH 7.5 containing 200 mM NaCl and 100 mM CaCl₂. Gels were stained for 30 minutes with 0.5% Coomassie brilliant blue R‐250 in 20% methanol and 10% acetic acid. Clear bands corresponding to areas of gelatinase activity were visualized against a blue background. Densitometry of active MMP2/9 bands was expressed as arbitrary units or fold changes versus basal conditions.

TEPMs were exposed to hypoxia for 24 h in the presence or absence of febuxostat, and supernatants were collected. Cell supernatants were clarified by brief centrifugation and concentrated using the Amicon Ultra-2, 3-kDa nominal molecular mass cutoff filters (Millipore, Billerica, MA, USA). The protein concentration of supernatants was determined by the BCA Protein Assay kit (Pierce, Thermo Fisher Scientific, Inc.). Equal amounts (5 μg/well) were loaded onto 10% zymogram plus (gelatin) protein gels (Novex; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for the gelatin zymogram or 10% SDS-PAGE containing 0.6 mg/mL rat type I collagen (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) for the collagen zymogram, and run for 90 min under a constant voltage of 125 V. Gels were incubated in a renaturing buffer (Novex; Thermo Fisher Scientific, Inc., Waltham, MA, USA), followed by incubation at 37 °C for 24 h in developing buffer (Novex; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Gels were stained with the SimplyBlue SafeStain (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Zymograms were analyzed by densitometry analysis using the ChemiDoc Touch Imaging System (BIO RAD Laboratories Inc., Hercules, CA, USA) (Supplementary Figure S2).