Anti s1pr1

Cells were seeded at 1.4x10 5 cells per well in a 12-well plate and grown as described above. Cells were transfected with siRNA using the TransIT-X2 System (Mirus, #MIR 600) according to the manufacturer's instructions. The following siRNAs were used: 50 nM β-arrestin-2 siRNA (Dharmacon) 5'-GGACCGCAAAGTGTTTGTG-3', 12.5 nM Dvl2 siRNA #2 (Qiagen, #SI00063441) 5'-CACGCTAAACATGGAGAAGTA-3', 25 nM of S1PR1 #1 (Qiagen, #SI00376201) 5'-ATGATCGATCATCTATAGCAA-3' and 25nM S1PR1 #2 (Qiagen, #SI00376208) 5'-TAGCATTGTCAAGCTCCTAAA-3' siRNAs or AllStars Negative Control siRNA (Qiagen, #1027281). After 72 h, protein expression was determined by immunoblotting using anti-β-arr2 A2CT (a generous gift from Dr. Robert Lefkowitz, Duke University), anti-Dvl2 (CST, #3216), anti-S1PR1 (Santa Cruz Biotechnology, #sc-25489), anti-GAPDH (GeneTex, #GTX627408), and anti-tubulin (CST, #86298S) antibodies.

EA.hy926 wildtype cells and EA.hy926 cells stably expressing PAR1-specific shRNA pSilencer Retro (Russo et al., 2009) (link) were grown in 10 cm dishes, serum-starved overnight, treated with 20 nM aPC, and lysed in Triton-X 100 lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM NaF, 1% Triton-X 100 supplemented with protease inhibitors). Cell lysates were homogenized, clarified by centrifugation and protein concentrations determined by BCA. Equivalent amounts of lysates were subjected to immunoprecipitations using the anti-S1PR1 (Santa Cruz Biotechnology, #sc-25489) and anti-PAR1 WEDE antibodies. Immunoprecipitates were resuspended in 2X LSB containing 200 mM DTT, and eluents immunoblotted using S1PR1 (Santa Cruz Biotechnology, # sc-25489), Cav-1 (CST, #610060), and PAR1 antibodies (Beckman Coulter, #IM2584) and developed by chemiluminescence.