Regulation of p38 MAPK by NO2-OA

The effect of the endogenous electrophilic lipid, NO₂-OA, on the activation and activity of p38 in vitro was examined. Purified recombinant p38α (0.1 μm) was incubated with NO₂-OA (10037, Cayman Chemical) in buffer containing 100 mm NaCl, 20 mm Tris, pH 7.5, 2 mm MgCl, and 200 μm ATP at 37 °C for 30 min. The reaction, in a 50-μl final volume, was started by the addition of 0.1 μg of MKK3b, 0.5 μg of ATF-2 fusion protein (9224, Cell Signaling) and incubated at 30 °C for 30 min. The reaction was stopped by the addition of 5× SDS sample buffer, and samples were resolved by SDS-PAGE and transferred onto PVDF membranes.
The effect of NO₂-OA on the ability of HePTP to dephosphorylate p38α was examined. Purified recombinant activated dually phosphorylated p38α (3 μm) was prepared as described previously (34 (link)) and incubated with NO₂-OA (15 μm) in buffer containing 5 mm MOPS, 10 mm NaCl, 10 μmm EDTA, pH 7.0, at 37 °C for 30 min. The reaction, in a 40-μl final volume, was started by the addition of 0.3 μm purified recombinant HePTP, prepared and purified from a plasmid vector kindly donated by W. Peti (39 (link)) and incubated at 30 °C for 30 min. The reaction was stopped by the addition of 5× SDS sample buffer, and samples were resolved by SDS-PAGE.