Anti phospho cpla2

The cells were scraped from the plates using RIPA Lysis Buffer (Merck Millipore, Darmstadt, Germany) containing a protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). After incubation on ice for 30 min, the cell lysates were centrifuged at 14,000 rpm for 20 min at 4°C. Proteins were quantified using BCA protein assay (Thermo Scientific, Waltham, MA, USA). The protein lysates were resolved on 10% sodium dodecyl sulfate polyacrylamide gels and then transferred to a polyvinylidene difluoride membrane. Anti-COX-2, anti-phospho-cPLA₂, anti-phospho-Lyn, anti-phospho-Syk, anti-phospho-Fyn, anti-phospho-PLCγ1, anti-phospho-ERK, anti-phospho-Akt, and anti-α-tubulin antibodies (Cell Signaling Technology, Boston, MA, USA) were used to detect COX-2, p-cPLA₂, p-Lyn, p-Syk, p-Fyn, p-PLCγ1, p-ERK, p-Akt, and α-tubulin, respectively. α-Tubulin was used as protein loading control. Blots were observed using a western blot detection kit (Thermo Scientific, Waltham, MA, USA). Protein bands were quantified using Image Lab software (Bio-Rad Laboratories, Richmond, CA, USA).

The cells were scraped from the plates with RIPA lysis buffer containing a protease and phosphatase-inhibitor cocktail (Roche, Basel, Switzerland). The lysates were quantified using the BCA protein assay kits (Thermo, MA, USA). Equal amounts of protein were resolved on 10% SDS-PAGE and then transferred to a PVDF membrane. Anti-COX-2, anti-β-actin, anti- phospho-Lyn, anti-phospho-Syk, anti-phospho-Fyn, anti-phospho-PLCγ1, anti-phospho-cPLA2, anti-phospho-ERK, and anti-phospho-Akt (Cell Signaling, MA, USA) were used to detect COX-2, β-actin and the phosphorylated form of Lyn, Syk, Fyn, PLCγ1, cPLA2, ERK, and Akt, respectively.

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.