Polyacrylamide ready gel

Corneal stromal tissues without epithelium were collected as mentioned in the above RT-qPCR methods. Frozen samples were homogenized in RIPA buffer (50 mM Tris base, 150 mM NaCl, 0.5% deoxycholic acid-sodium salt, 2% SDS, and 1% NP40, pH 7.5) containing 1x protease inhibitor cocktail (Sigma P8340). Cell lysates (20 µg) from each sample were separated on a 4–20% linear gradient Tris-HCl denaturing polyacrylamide Ready Gel (Bio-Rad) and transferred to PVDF membrane (Whatman). Membranes were blocked with 5% nonfat milk in TBST (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% Tween 20) and probed with primary antibody in the same buffer overnight at 4 °C. After three washes in TBST, membranes were probed with HRP-conjugated secondary antibody for an hour at room temperature and bond second antibody was further detected using an enhanced chemiluminescence assay (Supersignal West Pico, #34080; Thermo Fisher Scientific) and examined and photographed using a VersaDoc 4000MP imaging system (Bio-Rad). Antibodies are listed in Supplementary Material Table S2.

Frozen corneas isolated from experimental mice were homogenized in RIPA buffer (#89900 Thermo Fisher Scientific, Waltham, MA) containing 1× protease inhibitor cocktail (P8340, Sigma, St Louis, MO). Tissue lysates (20 µg) from each sample were separated on a 4% to 20% linear gradient Tris-HCl denaturing polyacrylamide Ready Gel (Bio-Rad Laboratories Inc., Hercules, CA) and transferred to PVDF membranes (Whatman, Maidstone, UK). The membranes were incubated with primary antibody overnight at 4°C and then were probed with horse radish peroxidase-conjugated secondary antibody for an hour at room temperature. The signal was detected using an enhanced chemiluminescence assay (Supersignal West Pico, #34080; Thermo Fisher Scientific); then examined and photographed using a VersaDoc 4000MP imaging system (Bio-Rad Laboratories Inc.). Antibodies used in this study were listed in Supplementary Table S2.