Fig. 5. Oxidant-sensitive cysteines are essential for p38 activation.

a The p38 MAPK pathway in C. elegans. Cysteines in pink were identified as redox-sensitive in our analysis, while cysteines in white were not responsive to H₂O₂ treatment. b Representative XICs showing changes in IPM-labeled peptides from SEK-1 (C213) and PMK-1 (C173). The profiles for light- and heavy-labeled peptide are shown in red and blue, respectively. The average R_T/C values calculated from two biological replicates are displayed below each XIC. c C213 (in red) in SEK-1 and C173 (in red) in PMK-1 are evolutionarily conserved and located close to the magnesium-binding DFG motifs (in blue). d, e Representative western blots in wild-type, SEK-1, and PMK-1 mutant animals carrying individual cysteine-to-serine mutations, and quantification showing that the two reactive cysteines are each important for t-BOOH-induced p38 phosphorylation (mean ± SEM, n = 3 experiments in (d); mean, n = 2 experiments in (e).) *P = 0.0318; ns, not significant (P = 0.3443); One-way ANOVA with Bonferroni post-test. Lysates were loaded onto two different gels for detection with different antibodies, and blots were processed in parallel. f Quantification of mRNA levels of the SKN-1 target gst-10 by qRT-PCR in wild-type, the PMK-1 C173S mutant (pmk-1(syb1415)), and the SEK-1 C213S mutant (sek-1(syb1398)) animals with or without 30 min t-BOOH treatment (mean, n = 2 experiments). g Survival curves of wild-type animals, and pmk-1(−)(km25), sek-1(−)(km4), PMK-1 C173S (syb1415), and SEK-1 C213S (syb1398) mutants in the presence of Pseudomonas aeruginosa PA14. n = 2 experiments with 280–388 worms per condition. Survival summary data are provided in Supplementary Table 1.