Phospho gsk 3β ser9

After release from quiescence in the presence of DMSO or GUTK, cells were harvested and treated with ice-cold RIPA lysis buffer supplemented with a protease inhibitor cocktail. Protein quantification, electrophoresis and western blotting were performed as previously described.^{42 (link)} Antibodies (human specific) used were c-MYC (Abcam, Epitomics, Cambridge, UK, #1472-1), S62-phospho-c-MYC (ab51156), T58-phospho-c-MYC (ab28842) and GAPDH (ab128915) were obtained from Abcam Company (Cambridge, UK). Additional antibodies were phospho-Rb (Ser807/811) (#9308), ERK1/2 (137F5) (#4695), phospho-ERK1/2 (Thr202/Tyr204) (#4370), GSK3β (27C10) (#9315) and phospho-GSK3β (Ser 9) (#9323) purchased from Cell Signaling Technology (Danvers, MA, USA), FBXW7 (A301-721A) were obtained from Bethyl Laboratories (Montgomery, TX, USA) and α-tubulin (sc-5286) from Santa Cruz Biotechnology (Dallas, CA, USA).
Immunoblot images were exported in the format of tagged image file and quantified using ImageJ 1.46 software (National Institute of Health). After normalization to loading control, the immunoblot bands of c-MYC and FBXW7 were plotted to determine the half-life. The 50% decrease in protein intensity based on the Y-axis was used to determine the corresponding value on the X-axis, which by definition is the time required for the proteins of interest to decrease to the half of the baseline levels.

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.