Fig. 6: Effects of MKP5 residue Y435 on MAPK engagement at the active site.

Full-length GST-tagged MKP5 WT, Y435A, C408S and C435/C408S mutants were transiently transfected with p38α MAPK-HA, JNK1-β-GFP into COS-7 cells for 48 h. Cells were harvested and lysed, followed by affinity purification with GSH Sepharose beads. a GST affinity complexes and whole cell lysates were immunoblotted with anti-HA, anti-GST, and phospho-p38 MAPK antibodies. b Densitometric quantitation of relative normalized p38α MAPK bound to GST-MKP5; c Relative normalized phosphorylated p38 MAPK in complex with MKP5; d GFP affinity complexes and whole cell lysates were immunoblotted with anti-GFP, anti-GST, and phospho-JNK antibodies. e Graph showing densitometric quantitation of relative normalized JNK bound to GST-MKP5; f Graph showing relative normalized phosphorylated JNK in complex with GST-MKP5. Data represent the mean ± SEM of four independent experiments. Statistical significance shown was generated using a two-way ANOVA.