Role of EGFR in LPA signal transduction. Subconfluent cell cultures were pre-treated with 1 μM gefitinib for 30 min before stimulation with 10 μM LPA for 1, 3 or 5 min as indicated. Cells were lysed for Western blotting or isoelectric focusing with NanoPro detection system as described in Methods. A: Western blots show phosphorylation of EGFR, Akt, ERK and p38 after LPA stimulation. The phosphorylation was inhibited by gefitinib pre-treatment in E10 and D2 cells. Cetuximab, in contrast to gefitinib did not inhibit Akt phosphorylation in D2 cells. In SCC-9 cells, Akt, p38, and ERK were phosphorylated by LPA, but not inhibited by gefitinib. No phosphorylation of EGFR was detected after LPA stimulation in the SCC-9 cells. As a positive control, EGFR phosphorylation after EGF stimulation was shown. n = 3. Graphs showing blot quantifications for E10 and D2 at 5 min are shown below the blots. B: NanoPro detection confirmed inhibition of LPA-induced ERK phosphorylation with gefitinib (1 μM) in E10 cells. Similar results were seen with cetuximab (0.16 μM). C: Western blots show tyrosine phosphorylation of EGFR and total tyrosine phosphorylation in SCC-9 cells after stimulation with EGF. No tyrosine phosphorylation is detected after LPA stimulation. n = 3 *indicates p ≤ 0.01.