Anti phospho raf

Purified CD4⁺ T cells were suspended in RPMI 1640 supplemented with 10% FCS, 2 mM glutamine and penicillin/streptomycin then left unstimulated or stimulated with 50 ng/ml PMA for 15 min at 37°C. Where indicated, cells were incubated in the presence or absence of Hyd (10 μM), H₂O₂ (50 μM), or ONOO⁻ (50 μM) for 60 min before PMA stimulation. Following stimulation the cells were lysed, then total and phosphorylated ERK, MEK, RAF and PKCδ measured by immunoblotting as described (16 (link)). The following primary Abs were used at a 1/1000 dilution: anti-phospho-raf (Ser³³⁸), anti-phospho-MEK1/2 (Ser^217/221), anti-MEK1/2 and anti-phospho-PKCδ (T⁵⁰⁵) (Cell Signaling Technology, Beverly MA). Polyclonal rabbit anti-active MAPK (1/5000) was purchased from Promega. Secondary Abs included anti-rabbit IgG horse radish peroxidase (HRP) (1/2000; Cell Signaling Technology) and anti-mouse IgG HRP (1/4000; Sigma-Aldrich).