P jnk 9255s

Platelets, megakaryocytes, and Dami cells were lysed using RIPA buffer (Thermo Fisher Scientific) for 30 min on the ice and then centrifuged at 12,000 × g for 10 min at 4°C. Equal amounts of sample were separated by SDS-PAGE (80~120 V for 1.5 h) and then transferred onto polyvinylidene difluoride membranes. The membranes were incubated with antibodies against GAPDH (5174S, Cell Signaling Technology), integrin alpha-IIb (CD41; ab33661, Abcam), p44/42 mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1/2 (ERK1/2; 4695S, Cell Signaling Technology), phospho-p44/42 MAPK ERK1/2 (4370S, Cell Signaling Technology), JNK (9252T, Cell Signaling Technology), p-JNK(9255S, Cell Signaling Technology), p-p38(9211S, Cell Signaling Technology), and p38 (9212S, Cell Signaling Technology) at 4 °C overnight after blocking of the membranes with 5% non-fat dry milk. All antibodies were diluted to 1:1000. The membranes were then incubated with goat anti-rabbit or anti-mouse antibodies (1:5000, Thermo Fisher Scientific) for 2 h at 25 °C. Protein bands were quantified using ImageJ software.