U46619

Mulberroside C (Figure 1) was provided by ChemFaces (Wuhan, China). Mouse monoclonal to CD62P (P-selectin) antibody was purchased from Biolegend (San Diego, CA, USA). Fura 2-AM (2-acetoxymethyl) and Alexa Fluor 488-conjugated fibrinogen were obtained from Invitrogen (Eugene, OR, USA). The BCA protein assay kit was obtained from Pierce Biotechnology (Rockford, IL, USA). The serotonin ELISA kit was provided by Labor Diagnostika Nord GmbH and Co. (Nordhorn, Germany). Collagen, U46619, and thrombin were purchased from Chrono-Log Co. (Havertown, PA, USA). The thromboxane B₂ assay kit, cAMP, cGMP enzyme immunoassay kit, and ATP assay kit were obtained from Cayman Chemical (Ann Arbor, MI, USA). Cell signaling (Beverly, MA, USA) supplied antiphospho-p38^MAPK, antiphospho-ERK (1/2), antiphospho-VASP (Ser¹⁵⁷), antiphospho-VASP (Ser²³⁹), antiphospho-cPLA₂ (Ser⁵⁰⁵), antiphospho-PI3K (Tyr⁴⁵⁸), antiphospho-Akt (Ser⁴⁷³), antiphospho-inositol-3-phosphate receptor type I (Ser¹⁷⁵⁶), antiphospho-PLC_γ2 (Tyr⁷⁵⁹), anti-β-actin, and antirabbit secondary antibodies.

VU0155069 (VU1) and VU0364739 (VU2) were purchased from Tocris Bioscience (Bristol, UK). Thrombin, collagen, and U46619 were purchased from Chrono-Log (Havertown, PA, USA). The fluorescein isothiocyanate (FITC)-conjugated anti-P-selectin antibody was purchased from Biolegend (San Diego, CA, USA). The anti-phospho-PLD1 (Thr¹⁴⁷), anti-phospho-p38 mitogen-activated protein kinase (MAPK) (Ser¹⁸⁰/Tyr¹⁸²), anti-phospho-p44/42 MAPK (extracellular signal-regulated protein kinases 1 and 2 [ERK1/2]) (Thr²⁰²/Tyr²⁰⁴), anti-c-Jun N-terminal kinase (JNK), and anti-phospho-Akt (Ser⁴⁷³) polyclonal antibodies (pAbs) and the anti-PLD2, anti-p38 MAPK, anti-p44/42 MAPK, anti-phospho JNK (Thr¹⁸³/Tyr¹⁸⁵), and anti-Akt monoclonal antibodies (mAbs) were purchased from Cell Signaling (Beverly, MA, USA). The anti-phospho-PLD2 (Tyr¹⁶⁹) pAb was purchased from Abcam (Cambridge, UK). The anti-PLD1 mAb was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The Hybond-P polyvinylidene difluoride membrane, an enhanced chemiluminescence (ECL) Western blotting detection reagent and analysis system, horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin G (IgG), and sheep anti-mouse IgG were purchased from Amersham (Buckinghamshire, UK). VU1 and VU2 were dissolved in dimethyl sulphoxide (DMSO) and stored at 4 °C until use.

Rhosin/RG04 was custom-synthesized as described [23 (link)]. Collagen (No. P/N 385), adenosine diphosphate (ADP) (No. P/N 384), adenosine triphosphate (ATP) standard (No. P/N 387), thrombin (No. P/N 386), and Chrono-lume^® (No. P/N 395) were obtained from Chronolog Corporation (Havertown, PA). Arachidonic acid (AA) (No. 90010) and U-46619 (No. 16450) were obtained from Chronolog Corporation (Havertown, PA). Chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or specifically noted sources. Anti-RhoA (67B9) antibody (No. 2117), Phospho-Myosin light chain 2 (Ser19) antibody (MLC) (No. 3671), Myosin light chain 2 (D18E2) antibody (No. 8505), and GAPDH (14C10) antibody (No. 2118) were purchased from Cell Signaling Technology (Danvers, MA, USA). Amersham ECL rabbit IgG, HRP-linked whole Ab (from donkey) (No. NA934) was ordered from Cytiva (Marlborough, MA, USA).