Figure 4. Prdx1 protects MKP-5 from oxidation-induced oligomerization and inactivation.

A and B. Flag-MKP-1 or Myc-MKP-5 were overexpressed together with untagged Prdx1 in 293T cells, using 1.0 μg of DNA. (Note: initial experiments included N-terminal tagged Prdx1 proteins. This enhanced slightly the MKP-1 oligomerization; we therefore used untagged Prdx1). Cells were treated for 30 min with increasing concentrations of H₂O₂ in serum-free medium, and protein lysates analyzed under non-reducing conditions by Western blotting for oligomerization of MKP-1 and MKP-5 (n= 3). C and D. As in A and B, with the exception of co-expressing untagged Prdx1-CI. (n=3) E-H, Western blot analysis of A-D for phosphorylation of p38MAPKα. Quantification include three independent experiments. Density of phosphorylated p38MAPKα Western blot bands were analyzed using Image J software (http://rsbweb.nih.gov/ij/) and normalized to densities of p38MAPKα protein bands. Lastly, values were normalized to density of untreated cells expressing exogenous Prdx1. P-values of p38MAPKα-phosphorylation for MKP-1 + WT-Prdx1: 0.011 (25μM H₂O₂), 0.002 (100μM H₂O₂), 0.004 (250μM H₂O₂). MKP-1 + CI-Prdx1: 0.01 (100μM H₂O₂). MKP-5 + WT-Prdx1: 0.04 (100μM H₂O₂), 0.013 (250μM H₂O₂). MKP-5 + CI-Prdx1: 0.014 (250μM H₂O₂). Western blots showing MKP-1 or MKP-5 co-expressed with Prdx1 WT or Prdx1 CI run under non-reducing conditions can be found in the supplemental information along with Western blot analysis under reducing conditions (Figs. 4SA and 4SB). I and J. 293T HEK cells were co-transfected with Flag-MKP-1 or Flag-MKP5 and with Prdx1 WT or Prdx1 CI, respectively, and treated with increasing amounts of H₂O₂ for 30 min in serum free medium. Lysates were run under reducing conditions, and analyzed by Western blotting for phosphorylation of p38MAPKα on Thr180 and Tyr182, expression of p38MAPKα, MKPs and Prdx1.