Fig. 8. Phosphorylation of caveolin is required for EGFR translocation but is dispensable for EGFR phosphorylation. Monolayers of caveolin-null cells were infected for 24 hours with virus containing wild-type caveolin or the caveolin Y14F mutant. Confluent fibroblast monolayers were treated with 50 µM of either S25 or P25 for 1 hour. (A) Cell layers were lysed in sample buffer. Lysates were electrophoresed, immunoblotted and analyzed for phosphorylated EGFR at Tyr845 (EGFR PY845). Blots were then stripped and re-probed with a total EGFR antibody to verify equal loading. (B) Western blots shown in A were scanned and the S25 value of EGFR PY845 seen in Cav^+/+ cells was set at 1. The bars show the range over two experiments. (C) Cells were then removed with EGTA treatment and SAM was isolated. Lysates were electrophoresed and immunoblotted for EGFR. Blots were then stripped and re-probed for paxillin. (D) Western blots shown in C were scanned and the EGFR value obtained from Cav^+/+ cells was set at 1. The bars show the range over two experiments.