Pro measure

Washed platelets were pre-incubated with experimental samples and stimulated for aggregation reaction as previously described. After terminating the reaction, lysates were then prepared by solubilizing and centrifuging platelets in a sample buffer (0.125 M Tris-HCl, pH 6.8; 2% SDS, 2% β-mercaptoethanol, 20% glycerol, 0.02% bromophenol blue, 1 μg/mL phenylmethylsulfonyl fluoride, 2 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin A). Protein concentration was determined by BCA assay (PRO-MEASURE; iNtRON Biotechnology, Seoul, Republic of Korea). Total cell proteins (30 μg) obtained from platelet lysates were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes in transfer buffer (25 mM Tris at pH 8.5, 0.2 M glycine, and 20% methanol). The membranes were blocked in TBS-T containing 5% nonfat dry milk and incubated with primary antibody diluted in a blocking solution. The membranes were then probed with antibodies of phospho-ERK2, ERK2, phospho-p38, p38, phosphor-PLCγ2, PLCγ2, phospho-PI3 K (p85), PI3 K and β-actin. The blots were then incubated with the horseradish peroxidase-conjugated secondary antibody. Antibody binding was visualized by enhanced chemiluminescence (iNtRON Biotechnology, Seoul, Republic of Korea).

Platelets were pre-incubated either with vehicle or different concentration of R. acetosa extract in the presence of 1 mM CaCl₂ at 37 °C for 2 min prior to stimulation with collagen (2.5 μg/mL) for 5 min under continuous stirring condition. Reacting was terminated by adding lysis buffer [0.125 M Tris–HCl, pH 6.8; 2% SDS, 2% β-mercaptoethanol, 20% glycerol, 0.02% bromophenol blue, 1 μg/mL phenyl methyl sulfonyl fluoride (PMSF), 2 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin A]. Proteins were quantified by BCA assay (PRO-MEASURE; iNtRON Biotechnology, Seoul, Korea) and total cell proteins (35 μg) from the lysates were segregated on 10% SDS-PAGE followed by transferring to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% skim milk and then probed with primary and secondary antibodies accordingly in 5% BSA solution. Finally, antibody binding was pictured by enhanced chemiluminescence (iNtRON Biotechnology, Seoul, Korea).

Platelet suspensions (3 × 10⁸/mL) were pre-incubated with MAE or vehicle (0.1% (v/v) DMSO) at 37°C for 2 min. Platelet activation was induced by the addition of 2.5 μg/mL collagen and the reaction was allowed to proceed for 5 min. After terminating the reaction, lysates were then prepared by solubilizing and centrifuging the platelets in sample buffer (0.125 M Tris-HCl, pH 6.8; 2% SDS, 2% β-mercaptoethanol, 20% glycerol, 0.02% bromophenol blue, 1 μg/mL phenylmethylsulfonyl fluoride (PMSF), 2 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin A). Protein concentration was determined using a BCA assay (PRO-MEASURE; iNtRON Biotechnology, Seoul, Republic of Korea). Total cell proteins (30 μg) from the platelet lysate were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes in transfer buffer (25 mM Tris, pH 8.5; 0.2 M glycine, and 20% methanol). The membranes were blocked in TBS-T containing 5% nonfat dry milk and incubated with primary antibody diluted in a blocking solution. The blots were then incubated with horseradish peroxidase-conjugated secondary antibody. Antibody binding was visualized using enhanced chemiluminescence (iNtRON Biotechnology, Seoul, Republic of Korea).