Fig. 3. Dermal fibroblasts were starved of growth factors in DMEM for 16 hours as described. 1-2x10⁶ cells were pre-incubated with either DMEM alone (0, 5-60 minutes ISO) or DMEM containing either 10 µM AG1478 (A,B) for 90 minutes or 10 µM PP2 for 6 hours at 37°C (C,D). Cells were either untreated (control, 0, 5-60 minutes ISO) or stimulated with DMEM containing inhibitor and 1 µM ß-AR agonist for 5-60 minutes at 37°C, unless otherwise noted. After treatment, cell lysates from each experiment were prepared and electrophoresed on the same 10% polyacrylamide gels and transferred to membranes. Membranes were immunoblotted with either an anti-ERK antibody, a anti-phospho ERK antibody (P-ERK) an anti-EGFR antibody (EGFR) or an anti-phosphotyrosine antibody (PY). Three blots from separate AG1478 or PP2 experiments were scanned for p-ERK or PY and densitometry performed using a gel plotting macro in NIH Image 1.62. Data was averaged, statistically analyzed and represented graphically (B,D,F). Values plotted are means ± s.e.m. (n=3). ^*P<0.01 between conditions and controls. # no significant difference between AG1478/control and AG1478/ß-AR agonist or PP2/control and PP2/ß-AR agonist. The data shown are representative of three independent experiments from three separate cell strains.