Western blot analysis of MKK3/6 Control Cell extracts using Phospho-MKK3 (Ser189)/MKK6 (Ser207) (22A8) Rabbit mAb #9236 (upper) and MKK3 Antibody #5674 (lower).
Boil for 3 minutes prior to use. Load 20 µl of phosphorylated and nonphosphorylated MKK3/MKK6 Control Cell Extracts per lane.
Supplied in SDS Sample Buffer: 62.5 mM Tris-HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v phenol red or bromophenol blue. Store at –20°C or at -80°C for long term storage.
Nonphosphorylated MKK3/MKK6 Control Cell Extracts: Total cell extracts from NIH/3T3 cells, serve as a negative control. Supplied in SDS Sample Buffer.
Phosphorylated MKK3/MKK6 Control Cell Extracts: Total cell extracts from NIH/3T3 cells, treated with 50 mJ UV light and a 30 minute recovery, serve as a positive control. Supplied in SDS Sample Buffer.
MKK3 and MKK6 are two closely related dual-specificity protein kinases that activate p38 MAP kinase (1-5). MKK3 and MKK6 both phosphorylate and activate p38 MAP kinase at its activation site, Thr-Gly-Tyr, but do not phosphorylate or activate Erk1/2 or SAPK/JNK. Phosphorylation of p38 MAP kinase dramatically stimulates its ability to phosphorylate protein substrates such as ATF-2 and Elk-1. MKK3 and MKK6 are both activated by different forms of cellular stress and inflammatory cytokines (4,5). Activation of MKK3 and MKK6 occurs through phosphorylation at Ser189 and Thr222 on MKK3 (2) and Ser207 and Thr211 on MKK6 (4,5).
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