Skip to main content
. 1998 Jul 21;95(15):8985–8990. doi: 10.1073/pnas.95.15.8985

Figure 1.

Figure 1

Effect of receptor tyrosine kinase inhibition on LPA-mediated signaling in Rat-1, COS-7, and L cells. (Left, Rat-1) Quiescent Rat-1 cells were pretreated with either AG1296 or AG1478 at the concentrations indicated and stimulated for 5 min with 1% BSA (control, CO), 10 μM LPA, 10 ng/ml EGF, or 30 ng/ml PDGF-BB as indicated. (Top) Lysates were immunoprecipitated (IP) with anti-EGFR (αEGFR) followed by SDS/PAGE and immunoblotting with anti-phosphotyrosine (αPY). The filter then was reprobed with αEGFR. (Middle) Lysates were immunoprecipitated (IP) with anti-Shc (αShc), and proteins were resolved by SDS/PAGE and immunoblotted with anti-phosphotyrosine (αPY). (Bottom) Lysates from Rat-1 cells were immunoprecipitated (IP) with anti-ERK (αERK). In vitro kinase activity of precipitated endogenous ERK with myelin basic protein (MBP) as substrate was assessed as described in Materials and Methods. Phosphorylated myelin basic protein was visualized by autoradiography. (Center, L cells) After pretreatment with solvent (DMSO) or with 1 μM AG1478, quiescent L cells were stimulated for 5 min with 1% BSA (control, CO), 25 μM LPA, 2 units/ml thrombin receptor peptide (TRP), UV light (100 J/m2 UV C), 10 ng/ml EGF, or 10 ng/ml PDGF-BB. An ERK mobility shift assay was used to detect ERK activation (see Materials and Methods). (Right, COS-7) Confluent serum-starved COS-7 cells were treated with agonists as above. (Top) Lysates from COS-7 cells transiently transfected with either vector alone or dominant negative EGFR (HER-CD533) were immunoprecipitated (IP) with anti-EGFR (αEGFR). After SDS/PAGE, immunoblotting with anti-phosphotyrosine (αPY) was performed. The filter was reprobed with anti-EGFR (αEGFR). (Middle) COS-7 cell lysates were processed as described under Rat-1, middle panel. Shc isoforms with different molecular weights are indicated on the right (p66, p52, p46). The filter was reprobed with anti-Shc (αShc). Two different exposures of the same blot are shown. (Lower) COS-7 cells were transfected with HA-ERK2, pretreated with AG1478, or control-treated. HA-ERK2 was immunoprecipitated with anti-HA (αHA), and kinase activity was assessed with an in vitro ERK assay (see Materials and Methods).