Fig. 6. P2Y₁R mRNA expression in VSMCs stimulated with UTP.

VSMCs isolated from WT or P2Y2R−/− mice were cultured in 12-well plates and grown in DMEM/10% FBS to at least 80% confluence. Cells were serum-starved overnight, followed by stimulation with 10 μM UTP. Total RNA was isolated, and reverse transcribed to cDNA using multiscribe reverse transcriptase and random primers. The mRNA relative abundance was determined by RT-PCR using P2Y₁R forward and reverse primers and the ΔΔCt method to calculate fold increase in mRNA levels for results from 6 independent experiments. P2Y₁R expression is calculated relative to GAPDH. There were no significant differences in the levels of P2Y₁R mRNA detected over time.