Anti rabbit igg horseradish peroxidase

G-Ro (molecular weight=957.1) was obtained from Ambo Institute (Daejon, Korea). Thrombin was obtained from Chrono-Log Corporation (Havertown, PA, USA). Anti-PI3K, anti-phosphor-PI3K p85 (Tyr⁴⁵⁸), anti-Akt, anti-phosphor-Akt (Ser⁴⁷³), anti-rabbit IgG-horseradish peroxidase, and lysis buffer were purchased from Cell Signaling (Beverly, MA, USA). Wortmannin (PI3K inhibitor) and miltefosine (Akt inhibitor) were purchased from Cayman Chemical (Ann Arbor, MI, USA). CytoSelect 48-well cell adhesion assay kits (fibronectin-coated, colorimetric format) were purchased from Cell Biolabs (San Diego, CA, USA). Eptifibatide (αIIb/β₃ inhibitor), GR 144053 (αIIb/β₃ inhibitor), and anti-β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyvinylidene difluoride (PVDF) membrane and enhanced chemiluminesence solution (ECL) were purchased from General Electric Healthcare (Amersham, Buckinghamshire, UK). Fibrinogen Alexa Fluor 488 conjugate was obtained from Invitrogen Molecular Probes (Eugene, OR, USA).

We purchased 20(S)-ginsenoside Rg3 from the Ambo Institute (Daejon, Korea) and thrombin and all materials for platelet aggregation from Chrono-Log Corporation (Havertown, PA, USA). We purchased all materials for buffer solution and Rp-8-Br-cAMPS, Rp-8-Br-cGMP, p-chlorophenylthio (CPT)-cAMP, 8-Br-cGMP from Sigma (St. Louis, MO, USA) and Fura 2-acetoxymethyl (AM) from Invitrogen (Eugene, OR, USA). Thapsigargin and cAMP/cGMP enzyme immunoassay kit were obtained from Cayman Chemical (Ann Arbor, MI, USA). Anti-IP₃-receptor type I, anti-phosphor-IP₃-receptor type I (Ser¹⁷⁵⁶), anti-extracellular signal-regulated kinase (ERK) (1/2), anti-phosphor-ERK (1/2), anti-rabbit IgG-horseradish peroxidase, and cell lysis buffer were obtained from Cell Signaling (Beverly, MA, USA). Anti-β-actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal to CD62P (p-selectin) antibody was purchased from Biolegend (San Diego, CA, USA). Polyvinylidene difluoride (PVDF) membrane and enhanced chemiluminesence solution (ECL) were purchased from General Electric Healthcare (Chalfont St. Giles, Buckinghamshire, UK).

Heart tissue was homogenized in RIPA Buffer (50 mmol/L Tris pH 7.4, 150 mmol/L NaCl, 1% Triton X‐100, 0.5% Na deoxycholate, 0.1% SDS, 1 mmol/L EDTA, and 1X Complete Mini EDTA‐free protease inhibitor; Roche, Indianapolis, IN). For in vitro experiments, cells were scraped in RIPA buffer. Samples were sonicated using a probe sonicator. Debris was removed by centrifugation. Protein levels were quantified using a BCA assay (ThermoFisher Scientific Pierce, Rockford, IL). Ten micrograms of protein were size separated using SDS‐PAGE, transferred to nitrocellulose membranes, and subjected to standard immunoblotting procedures using antibodies targeted against G9a/Ehmt2 (C6H3), H3K9me2 (D85B4), Histone H3 (D1H2), Beta Tubulin (9F3), Actin, and mTOR (7C10) according to the manufacturer's instructions (Cell Signaling, Danvers, MA). Anti‐rabbit IgG‐horseradish peroxidase (Cell Signaling) was used as a secondary antibody. Enhanced chemiluminescent detection (Amersham GE Healthcare Life Sciences, Pittsburgh, PA) was used to detect the signal. Stripping of membranes were performed with Restore^™ Western Blot Stripping Buffer (Thermo Scientific, Grand Island, NY). The background‐subtracted integrated intensity was quantified for each protein of interest relative to the loading control using ImageJ software (US National Institutes of Health).