Effects of LPA and Ki16425 on ERK1/2 phosphorylation assessed by isoelectric focusing with NanoPro 1000. Subconfluent cell cultures of E10 cells were untreated or pre-treated with Ki16425 for 30 min, and then stimulated with 10 μM LPA and lysed for isoelectric focusing with NanoPro detection system as described in Methods. A: Typical pI spectra. Total ERK was detected in control (upper left) and LPA-stimulated E10 cells (upper right). Peaks were verified with a phospho-ERK antibody in control (lower left) and LPA-stimulated cells (lower right). B: ERK phosphorylation in E10 cells in response to LPA with or without Ki16425 before LPA stimulation. n = 3. Bars represent mean ± SEM. *indicates p = 0.018.