Fig. 2. RT-PCR analysis of LPA receptor expression and LPA-stimulated activation. (A) Total RNA was prepared from PANC-1, BxPC-3 and MiaPaCa-2 cells and used as template for RT-PCR using specific oligonucleotides for LP_A1/Edg2, LP_A2/Edg4 and LP_A3/Edg7. The amounts of cDNA used as template were adjusted to similar levels according to the amount of a -actin fragment present in the samples determined by semi-quantitative PCR. A sample containing no single-stranded cDNA was analyzed for comparison (Control). The amplified DNA fragments were fractionated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. The sizes for LPA receptor PCR-products are: LP_A1/Edg2: 349 bp; LP_A2/Edg4: 798 bp; LP_A3/edg7: 382 bp. (B) LPA-stimulated [³⁵S]GTP[S] binding to cell membranes. PANC-1 and BxPC-3 cells were incubated for 16 hours prior to membrane preparation in DMEM containing 10% FCS with or without PTX (50 ng/ml). Binding of [³⁵S]GTP[S] to 6 µg of membrane protein was assayed after addition of LPA at the indicated concentrations for 2 hours at 30°C in assay buffer containing 100 mM NaCl and 10 µM GDP. Fold stimulation of [³⁵S]GTP[S] binding as compared to membrane preparations treated without ligand is expressed as mean values ± s.e.m. (n=4).