Figure 5.

LPA signaling to ERK in L cells involves PDGFR. Confluent 15-cm dishes were serum-starved for 16 h and stimulated with 1% BSA (control, CO) (10 min), 3 ng/ml PDGF-BB (10 min), or 25 μM LPA (indicated time points). Lysates were immunoprecipitated with anti-PDGF-β-R (αPDGF-β-R) followed by SDS/PAGE and immunoblotting with anti-phosphotyrosine (αPY) (Left). In a similar experiment, the filter was reprobed with anti-PDGF-β-R (αPDGF-β-R) (Right) (A). Quiescent L cells were preincubated for 10 min with DMSO or 10 μM AG1296. Tyrosine phosphorylation of the precipitated PDGF-β-R was determined by immunoblotting with anti-phosphotyrosine (αPY) (B). L cells were transfected transiently with HA-ERK2 and either vector alone or a dominant negative PDGF-β-R. The cells were serum-starved for 16 h and stimulated (5 min) before lysis with 1% BSA (control, CO), 10 ng/ml PDGF-BB, or 25 μM LPA. Lysates were immunoprecipitated with anti-HA (αHA) and subjected to SDS/PAGE. ERK activation was detected by immunoblotting with a phopho-specific anti-ERK (αPY-ERK) that only detects tyrosine-phosphorylated activated ERKs (C).