Polyclonal rabbit anti active mapk

Purified CD4+ T cells were suspended in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine and penicillin/streptomycin and then left unstimulated or stimulated with 50 ng/ml phorbol myristate acetate (PMA) for 15 minutes at 37°C. Where indicated, cells were incubated in the presence or absence of hydralazine (10 μM), H₂O₂ (50 μM), or ONOO⁻ (50 μM) for 60 minutes before PMA stimulation. Following stimulation, the cells were lysed, then total and phosphorylated ERK, MEK, RAF, and PKCδ were measured by immunoblotting as described (16 (link)). The following primary antibodies were used at a 1:1,000 dilution: anti–phospho-Raf (Ser³³⁸), anti–phospho–MEK-1/2 (Ser^217/221), anti–MEK-1/2, and anti–phospho-PKCδ (T⁵⁰⁵) (Cell Signaling Technology). Polyclonal rabbit anti-active MAPK (1:5,000) was purchased from Promega. Secondary antibodies included anti-rabbit IgG horseradish peroxidase (HRP; 1:2,000) (Cell Signaling Technology) and anti-mouse IgG HRP (1:4,000; Sigma-Aldrich).