Mkk3b

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.

Antibodies against phosphorylated p38, phosphorylated MKK3/6, MKK6, and MKK3b were from Cell Signaling Technology (Beverly, MA, USA). Antibodies against p38, HA, GFP, and Actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against RACK1 was from BD Biosciences (Franklin Lakes, NJ, USA). Glutathione-Sepharose beads, Protein A-Sepharose beads, Hanks’ balanced salt solution (HBSS), collagenase I, PI, hoechst (H33258), cycloheximide, and antibody against FLAG were from Sigma-Aldrich (St. Louis, MO, USA). p38 inhibitors SB203580 and SB239063 were from CalBiochem (San Diego, CA, USA). ECL chemiluminescence kit was from Amersham (Arlington Heights, IL, USA). In vitro protein translation system was from Promega (Madison, WI, USA).