2.1 |. Materials

Chemicals and vendors were as follows: fetal bovine serum, antibiotic–antimycotic solution, sodium pyruvate, L-glutamine were purchased from Gibco (Gaithersburg, MD); Dulbecco’s modified Eagle’s medium (DMEM), ProLong Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI), and siPORT™ Amine Transfection Agent were from Thermo Fisher Scientific (Waltham, MA); Duolink® proximity ligation assay kit, penicillin–streptomycin, and trypsin-ethylenediaminetetraacetic acid (EDTA) solution, anti-P2Y13, horseradish-peroxidase (HRP)-conjugated anti-mouse IgG, and HRP-conjugated anti-rabbit IgG, phosphatase inhibitor cocktail, and Ado were obtained from Sigma-Aldrich (St. Louis, MO). Endothelial basal medium-2 (EBM-2) and EGM™-MV BulletKit™ were obtained from Clonetics (San Diego, CA). Dynabeads® Protein G were from Life Technologies (Grand Island, NY). Protease inhibitor cocktail, Set III, and anti-β-tubulin were obtained from EMD Millipore (Darmstadt, Germany). X-tremeGENE HP DNA transfection reagent was from Roche (Indianapolis, IN). Four to twenty percent of Mini-PROTEAN® TGX™ Gel, 0.2-μM pore size polyvinylidene fluoride (PVDF) membrane, Laemmli sample buffer and Trans-Blot® Turbo™ Transfer System, iScript™ cDNA synthesis kit, and SYBR Green were purchased from Bio-Rad (Hercules, CA). Enhanced chemiluminescence (ECL) reagent (Pierce, Rockford, IL). TRIzol reagent was from Life Technologies. Polyclonal antibodies for dual MYPT1 phosphorylation (pMYPT1^{Ser696/Thr697}) were generated and affinity-purified as previously described (Sutherland, MacDonald, & Walsh, 2016). Anti-ppMLC^T18/S19, anti-PKAcα, and anti-EPAC1 were from Cell Signaling Technology (Beverly, MA). AKAP2 antibody was from Bethyl Laboratories Inc. (Montgomery, TX). Anti-c-myc, anti-HA antibody, rabbit anti-P2Y4-antibody, mouse anti-G_i2-antibody, normal rabbit serum, nonspecific (scrambled) small interfering RNA (siRNA), EPAC1, P2Y4, and P2Y13 siRNA were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). AKAP2, P2Y14, and PKAcα specific siRNA duplexes were from Dharmacon Research (Lafayette, CO). P2Y1, P2Y11, and P2Y12 receptors specific siRNAs were obtained from Ambion (Austin, TX). Anti-P2Y2, anti-P2Y12, and anti-P2Y14 antibody were from Abcam (Cambridge, MA). Anti-P2Y1 and anti-P2Y11 antibody were from Alomone Labs (Jerusalem, Israel). HyBlot® ES autoradiography film was from Denville Scientific Inc. (Holliston, MA). ATPγS was purchased from Tocris (Mineapolis, MN). cAMP assay EIA kit was from Cayman Chemicals (Ann Arbor, MI). PKA activity assay kit was from Enzo Life Science (Farmingdale, NY).