Functional effects of SPGF are blocked by ErbB inhibitors. (A) Inhibition of human foreskin fibroblast proliferation by ErbB inhibitors. Human fibroblasts were treated and processed as described in Methods. Percentage of cells entering S phase is plotted. Gray area represents the baseline S phase after serum starvation. (B) Inhibitors block cellular protein tyrosine phosphorylation triggered by SPGF. HeLa cells were pretreated with 50 nM inhibitors (PP2, 10 μM) at 37°C for 30 minutes and then stimulated with 50 ng/ml SPGF at 37°C for 15 minutes. Minus (–) indicates no inhibitors or SPGF, and plus (+) indicates SPGF addition only. Total cell lysates were analyzed by Western blotting (WB) with 4G10 anti-phosphotyrosine (P-Y) mAb. Arrows indicate positions of phosphorylated substrates affected by ErbB-1 inhibition. Numbers on left side of the panel indicate the molecular marker in kDa. (C) ErbB-1 internalization prevented by inhibitors. A431 cells were pretreated and processed as described in Methods. MFI is recorded by FACS. Results are representative of 3 independent experiments. The inset shows the ErbB-1 cellular distribution pattern in HeLa cells in the absence (–) or presence of SPGF. (D) Inhibitors block association of ErbB-1 and c-Cbl and subsequent ErbB-1 degradation. HeLa cells were pretreated with inhibitors and stimulated (37°C, 5 minutes) with SPGF as above. Total cell lysates were immunoprecipitated (IP) with anti-EGFR or anti–c-Cbl and subjected to Western blot with either antibody.