Quantitative Western Blot Analysis of Proteins

Protein was extracted from cells and posterior eyecup without conjunctiva, sclera, and muscle tissue with RIPA buffer containing protease inhibitor. The concentration of soluble protein was measured using BSA as standard (SpectraMax iD5, Molecular Devices, Sunnyvale, CA, USA). Equal amounts of protein (30 μg) were resolved on TGX-precast gels (Bio-Rad Laboratories Inc., Hercules, CA, USA) and transferred to PVDF blotting membranes (Millipore, Billerica, MA, USA). Membranes were probed with respective primary antibodies overnight at 4 °C (The antibodies and dilutions used are listed in Table 2). After incubation with the appropriate secondary antibodies (Vector Laboratories, Burlingame, CA, USA), protein bands were visualized by a chemiluminescence (ECL) detection system (Bio-Rad Laboratories Inc., Hercules, CA, USA). Equal protein loading was confirmed with GAPDH. Protein band intensity was measured by Image J software (Version 1.51 23 April 2018 and URL accessed on 12 April 2018) (https://imagej.nih.gov/ij/). The quantification indicates the relative amounts as a ratio of each protein band to the appropriate loading control and expressed arbitrary units [35 (link)].