Total syk

PMQ (3,3′,4′,5,7-pentamethylquercetin, HPLC grade with ≥98% purity; Fig. 1) was synthesized at the laboratory of Prof. Jin in our department31 (link). Thrombin and U46619 were obtained from Sigma (St. Louis, MO, USA). Collagen and luciferin-luciferase was purchased from Chrono-Log Corp (Havertown, PA, USA). Antibodies against total JNK, phospho-JNK (Thr183/185), total-AKT, total-Syk, total-PLCγ2, total-Erk, phospho-Erk (Tyr 204), total-GSK3β were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phospho-p38 (Thr180/Tyr182), phoshpo-PLCγ2 (Tyr1217), phospho-Syk (Tyr525/526), phospho-AKT (Ser473), and phospho-GSK3β (Ser9) were obtained from Cell Signaling (Beverly, MA, USA). Anti-CD62P (P-selectin) antibodies were obtained from BD Biosciences (San Jose, CA, USA). The ECL western blotting detection reagent was obtained from Pierce Chemical Co (Rockford, Illinois, USA). 2.5 mM, 5 mM, 10 mM PMQ stock solution was dissolved in 80% dimethylsulfoxide (DMSO) and stored at room temperature until use, 1 μl stock solution was further diluted in 250 μl platelet suspension in vitro experiment. And PMQ was dissolved in dilution buffer ( polyethyleneglycol: Glycerin: physiological saline = 4 : 2 : 9) in vivo experiment.

Pimpinellin (HPLC≥98%) was purchased from Shanghai Macklin Technology Corp (Macklin, Shanghai, China). Luciferin and collagen were purchased from the Chrono-Log Corp (Havertown, PA, United States). Thrombin, ADP, and FITC-Phalloidin were purchased from Sigma (St. Louis, MO, United States). PAC-1 and CD62P (P-selectin) antibody were purchased from BD Biosciences (San Jose, CA, United States). Antibody against total-p85, total-Akt, total-GSK3β, total-p38, total-ERK, total-JNK, total-Syk, total-SLP76, total-PLCγ2, and phospho-Akt (Ser473) were from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Antibody for phospho-PI3K (p85/p55), phospho-GSK3β, phospho-p38, phospho-ERK, phospho-JNK, phosphor-SLP76, phosphor-PLCγ2, and phospho-PKC substrates was from Cell Signaling Technology (Beverly, MA, United States). The phospho-Syk antibody was obtained from GeneTex International Corp (GeneTex, Taiwan, China). GAPDH was purchased from Proteintech Group, Inc (Proteintech, IL, United States). The thromboxane B2 and cyclic adenosine monophosphate (AMP) enzyme immunoassay (EIA) kits were from Cayman (Ann Arbor, MI, United States); ECL Western blotting detection reagent was obtained from Millipore Corp (Millipore, MA, United States). Pimpinellin was dissolved in DMSO and stored at −20°C.