Fig. 1. Stable expression of human LPA₁ in HeLa cells and LPA stimulation of ERK1/2 activity. (A) Cell extracts were prepared from either untransfected HeLa cells (H) or from stably transfected HeLa cells expressing LPA₁ (E2). Various amounts of extracts were separated by 10% SDS-PAGE, transferred to nitrocellulose and probed with rabbit anti-FLAG antibodies and processed for chemiluminescence detection. A single band of approximately 43 kDa was detected by anti-FLAG antibodies in extracts prepared from stably transfected LPA₁-expressing cells, but not from untransfected HeLa cells. (B) Stable LPA₁-expressing HeLa cells or untransfected HeLa cells were stimulated with 10 µM LPA for the indicated times and washed at 4°C prior to detergent solubilization. Equal amounts of cell extracts (30 µg) were separated by SDS-PAGE and probed with rabbit antibodies against dually phosphorylated ERK1/2 or total ERK1/2 as described in the Materials and Methods. LPA-stimulated ERK activity is maximal between 5 and 10 minutes and then declines by 30 minutes in the FLAG-LPA₁ stable transfectants.