Anti tyr 416

Levels of total and phosphorylated c-Src (anti-Tyr-416, Cell Signaling Technology; pan-c Src, Proteintech), Syk (anti-Tyr-525 and pan-Syk, Bioworld Technology), PLCγ2 (anti-Tyr-1217 and pan-PLCγ2, Bioworld Technology), AKT (anti-Thr308 and anti-Ser473, Cell Signaling Technology; pan-AKT, Affinity Biosciences), or GSK-3β (anti-Ser9, Cell Signaling Technology; pan-GSK-3β, Affinity Biosciences) were assessed by SDS-PAGE/western blot. The density of protein band was quantified using Image J software and the phosphorylation level was shown as a ratio to the total level.

Human platelets were treated with CRP (5 μg/ml) or thrombin (1 U/ml) in the presence of different doses of matrine or vehicle for 5 min followed by measuring AKT (anti-Thr308 and anti-Ser473, Cell Signaling Technology; pan-AKT, Affinity Biosciences), ERK1/2 (anti-Thr202/Tyr204 and pan-ERK1/2, Cell Signaling Technology), p38 MAPK (anti-Thr180/Tyr182, Cell Signaling Technology), VASP (anti-Ser157, Affinity Biosciences; anti-Ser239 and pan-VASP, Cell Signaling Technology) by SDS-PAGE/western blot. The density of protein band was quantified using Image J software and the phosphorylation level was shown as a ratio to the total level.
For some experiments, the levels of c-Src (anti-Tyr-416, Cell Signaling Technology; pan-c Src, Proteintech) was measured in platelets after spreading on fibrinogen or bovine serum albumin (as a control), or under the condition of clot retraction (platelets were stimulated with thrombin (1 U/ml) in the presence of 2 mM Ca²⁺ and 0.5 mg/ml fibrinogen).