Fig. 6. Caveolin is required for uPAR-dependent EGFR translocation to the focal adhesion. Cells were transfected with EGFR siRNA (A-C) or caveolin siRNA (D, and see Fig. 2) as described in the Materials and Methods. (A) Cells treated with EGFR siRNA or non-targeting siRNA were lysed in sample buffer under reducing conditions, electrophoresed and analyzed by western blot for EGFR. Blots were stripped and re-probed for Erk2 as a loading control. (B) Blots shown in A were scanned and data was normalized against Erk2. Values from cells receiving control siRNA were set at 1. Bars reflect the range of knockdown (n=2). (C) Cells treated with control siRNA or EGFR siRNA were incubated with 50 µM P25 or S25 for 1 hour. SAMs were isolated by removing cells from the substrate with EGTA treatment and solubilized in sample buffer under reducing conditions. SAMs were electrophoresed, immunoblotted and analyzed for caveolin phosphorylated at Tyr14. Blots were stripped and re-probed for paxillin. (D) SAMs were prepared from cells treated with control siRNA or caveolin siRNA and electrophoresed, immunoblotted and analyzed for EGFR. Blots were stripped and re-probed for paxillin.