Quantitative Protein Analysis by Western Blotting

For western blotting, cell lysates were generated by resuspending cells in RIPA buffer (Sigma), then sonicated as previously described (Hearst et al., 2010 (link)). Lysates were subjected to sodium dodecyl sulfate– polyacrylamide gel electrophoresis (4–20% acrylamide precast gels; Bio-Rad Laboratories, Hercules, CA, United States) as described earlier (Hearst et al., 2010 (link)). Equal amounts of proteins were loaded in each well. Proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad), blocked for 1 h with blocking solution (1X TBS, 0.1% Tween-20 with 5% w/v non-fat dry milk) and incubated overnight with primary antibodies: Casp-3 and βIII Tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, United States), GFAP and ACTB (Cell Signaling Technology, Danvers, MA, United States). Western blots were imaged using Alexa-794 secondary antibodies (Santa Cruz) and the Odyssey Infrared Imaging System (LI-COR Biosciences, NE, United States). Western blots were analyzed using Image J software, as previously described (Hearst et al., 2010 (link)). The average ratios from protein band optical densities were obtained from three independent experimental repeats.