Mouse anti p erk1 2 tyr 204

Lipopolysaccharide derived from Escherichia coli O111:B4, TRAP-6, ADP and prostaglandin (PG) E1 were obtained from Sigma-Aldrich (St. Louis, MO, United States). Pam3CSK4 was purchased from InvivoGen (San Diego, CA, United States) and thrombin from Biopool (Umea, Sweden). Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human P-selectin (CD62P), anti-CD40L (CD154), and CD63 and PE-conjugated mouse anti-human CD41, TLR2, and TLR4 were from BD Biosciences (San Jose, CA, United States). FITC-conjugated CD45 and Human CCL5 (RANTES) ELISA Max were purchased from BioLegend (San Diego, CA, United States) and Quantikine ELISA Human P-selectin/CD62P was from R&D Systems (Minneapolis, MN, United States). Rabbit anti-human VWF and HRP-conjugated anti-human VWF were from Dako (Glostrup, Denmark), mouse anti- p-ERK1/2 (Tyr 204) and rabbit anti ERK 1/2 were obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Neutrophils (6 × 10⁶) were incubated with or without PMA for 45 minutes at 37 °C, lysates were prepared with RIPA buffer and 40 μg proteins were resolved by SDS-PAGE. Immunoblotting was performed with mouse anti- p-ERK1/2 (Tyr 204) (Santa Cruz Biotechnology, Dallas, TX, USA) and protein loading was assessed with rabbit anti-ERK1/2 followed by the corresponding HRP-conjugated secondary antibodies and detection by enhanced chemiluminiscence.