Western Blot Analysis of Cellular Signaling

Wounded HCEC were dissolved in Laemmli’s buffer with 1% SDS, 5% 2-mercaptoethanol (Life Technologies), and proteinase inhibitors (Sigma-Aldrich, St. Louis, MO). For Western blot analysis, 8% to 16% gradient Tris-glycine SDS polyacrylamide gels were used (Life Technologies). Gel loading was normalized using monoclonal mouse antibodies AC-74 to β-actin or TUB 2.1 to β-tubulin (Sigma-Aldrich). After transfer to nitrocellulose membranes, blots were blocked in 5% defatted milk and incubated with primary antibodies, rabbit anti-p-EGFR (44-784G, Life Technologies), mouse anti-EGFR (2239, Cell Signaling Technology, Danvers, MA), mouse anti-p-p38 (ab50012, Abcam, Cambridge, MA), rabbit anti-p38 (9212, Cell Signaling) or rabbit anti-pAkt (9271, Cell Signaling). Alkaline phosphatase–conjugated (Jackson ImmunoResearch Laboratories, West Grove, PA) or horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, Texas) were used with their standard substrates BCIP/NBT (Life Technologies) or Pierce ECL Western Blotting substrate (Thermo Fisher Scientific, Rockford, IL), respectively.