Western blot analysis were performed as previously described.
16 Protein from cultured cells was isolated, and protein concentration was determined with a BCA Protein Assay (Bio-Rad, Hercules, CA). To examine kinase activation, the cells were either treated with 50 ng/mL PDGF (PeproTech, Rocky Hill, NJ) for 30 minutes or incubated on a collagen I–coated plate for 2 hours before protein isolation. Where indicated, AKT phosphorylation was blocked by pretreating cells for 1 hour with 50 μM of the small molecule inhibitor Ly294002 (Cell Signaling Technology, Danvers, MA). A total of 15 μg of protein was loaded in each lane and then fractionated by 4% to 20% SDS-PAGE gradient gel under reducing conditions. Each cell lysate was examined in triplicate. The membrane was then blocked with nonfat milk in TBS Tween (Upstate, Charlottesville, VA). Blots were incubated overnight with primary antibody at a dilution of 1:500 for PMP22, 1:50 for p-FAK, 1:1000 for p-AKT, and 1:5000 for β-actin. Horseradish peroxidase–conjugated goat anti-rabbit or horseradish peroxidase–conjugated goat anti-mouse was exposed to the blots at a 1:2000 dilution. Blots were then developed with enhanced chemiluminescence to visualize bound antibody (Pierce, Rockford, IL) and were quantified using β-actin as an internal control. Western blot analyses were quantified using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at
http://rsb.info.nih.gov/ij/index.html). The blots were digitized using a flatbed scanner, and the band density was measured using Image J. To account for loading variability, β-actin was used to normalize each sample. At least three independent experiments were performed, and, where indicated, the results were evaluated for statistical significance using a Student's
t-test (unpaired, one-tailed). A level of
P < 0.05 was considered statistically significant.