Figure 3.

LPA stimulates p130Cas phosphorylation through LPA receptors 1 and/or 3. (A) Quantification of the mRNA levels of LPA receptors via quantitative RT-PCR in HeyA8, OVCA429, and SKOV3 cells. (B) Effect of LPAR inhibitor KI16425. OVCA429 cells were plated and allowed to grow overnight. The following day, the cells were serum starved for 16 hours and then pretreated with the indicated dosages of KI16425 for 15 minutes. Following KI16425 pretreatment, the cells were then stimulated with 20 µM of LPA for 20 minutes and then lysed. Immunoblot analysis was carried out with the lysates using antibodies for Tyr-410 p130Cas. The blot was stripped and reprobed for total p130Cas levels. GAPDH was checked to ensure equal loading of each lane. (C) Effect of the LPAR1/3 inhibitor VPC32183. HeyA8, OVCA429, and SKOV3 cells were plated and allowed to adhere overnight. The following day, the cells were serum starved for 16 hours and then pretreated with the indicated dosages of VPC32183 for 15 minutes. After 15-minute pretreatment with VPC32183, the cells were treated with 20 µM of LPA for 20 minutes and subsequently lysed. The lysates were subjected to immunoblot analyses using antibodies to Tyr-410 p130Cas or Tyr-419 Src and then stripped and reprobed for total p130Cas and Src. The level of GAPDH was checked to ensure equal loading of each lane.