Fig. 2. Recruitment of substrates to the PDGFβR in HDFs expressing the different E5 proteins. (A) HDFs stably expressing the indicated E5 protein, v-Sis or no viral oncogene (control) were lysed after growing to 85-95% confluence, and cell extracts were immunoprecipitated (IP) with a PDGFβR antiserum. PDGFβR (PR) IPs were subjected to immunoblotting for p85-PI3K (PI3-K), SHP2, PLC-, p120RasGAP or Grb2. The uppermost portion of each blot was probed for PDGFβR to determine the amount of total receptor that was immunoprecipitated. For each substrate, a representative blot from several different experiments is shown. (B) The relative band intensities of the indicated substrates that co-immunoprecipitated with the PDGFβR as depicted in A were quantitated using the Image J program and normalized for total receptor amounts. Each normalized value was converted to a percentage relative to the normalized value for the wild-type E5-expressing HDFs, and the mean percentage from several different experiments with standard error is shown. This illustrates the amount of substrate bound to the PDGFβR relative to that in the wild-type E5-expressing HDFs.