Figure 1.
LPA induces the tyrosine phosphorylation of p130Cas. (A) Antibody array analysis indicated that p130Cas is tyrosine phosphorylated in an LPA-dependent manner. The dark spots, in decreasing order of magnitude, at D4, E2, F1, and H3 represent the tyrosine phosphorylation of p130Cas, FAK, CDC25A, and integrin β3, respectively. HeyA8 cells were plated at 5 × 105 per plate, allowed to adhere and grow overnight, serum starved for 16 hours and either left untreated or treated with 20 µM of LPA for 20 minutes, and then lysed with RIPA buffer. The lysates were incubated with an antibody array for 2 hours at room temperature, washed, and then incubated with an HRP-conjugated pan-tyrosine at 4°C overnight. After 16 hours, the antibody array was washed and developed. (B) Immunoprecipitation of p130Cas confirmed the antibody array data that p130Cas is tyrosine phosphorylated in an LPA-dependent manner in ovarian cancer cells. Lysates from HeyA8, OVCA429, or SKOV3 cells were subjected to immunoprecipitation using an antibody specific to p130Cas. Immunoprecipitates containing p130Cas were resolved by 10% SDS-PAGE, and immunoblot analysis was carried out using the same antibody specific to phosphotyrosine as used in the antibody array. The blot was then stripped and analyzed for total p130Cas levels.