Fig. 3. Cholesterol depletion and caveolin knockdown inhibit uPAR-mediated EGFR phosphorylation. (A,B) Confluent fibroblast monolayers were incubated with MβCD (10 mM) for 30 minutes. Cells were washed and incubated with 50 µM of the uPAR ligand, P25, or the control peptide, S25. (A) Cells were fixed and incubated with mAb74, a monoclonal antibody directed against an activated conformation of EGFR, for 1 hour. Bound antibody was detected by ELISA. n=3; error bars indicate s.e.m. Asterisk (*) indicates data that is significantly different from P25 treatment alone (t-test, P<0.05). (B) Cell layers were lysed in gel sample buffer under reducing conditions. Lysates were electrophoresed and immunoblotted using an antibody against the EGFR phosphorylated at Tyr845 (EGFR PY845). The blot was then stripped and re-probed with a total EGFR antibody to assure equal loading. (C,D) Cells were transfected with caveolin siRNA or a non-targeting siRNA for 72 hours. Cells were then incubated with 50 µM S25 or P25 for 1 hour. Cell layers were lysed in sample buffer. (C) Lysates were electrophoresed and immunoblotted using an antibody against EGFR PY845. The blot was then stripped and re-probed with a total EGFR antibody to assure equal loading. (D) Lysates were electrophoresed and immunoblotted in parallel using an antibody against Src family kinases phosphorylated at Tyr418 (Src PY418) or a pan Src antibody to detect total Src. The arrowhead indicates Src.