Figure 3.

JNK2 activity is reversibly inhibited by H₂O₂. A, JNK2 kinase assay. FLAG-JNK2α2–expressing chondrocytes were treated with FN-f to stimulate JNK phosphorylation. Cellular proteins were reduced prior to affinity capture of JNK2 with anti-FLAG magnetic beads. A single immunoprecipitation (IP) was then split into multiple fractions and treated or not with various amounts of H₂O₂ for 10 min prior to the addition of catalase to remove excess H₂O₂. Protein kinase A activity was then initiated by the addition of prereduced c-Jun and ATP, and JNK2 activity was measured as a function of c-Jun phosphorylation. B, WT FLAG-JNK2 was immunoprecipitated from 60 μg of lysate and then split into multiple tubes prior to treatment with 0, 0.5, 1, 3, or 20 μm H₂O₂ for 10 min, and kinase activity was measured as described in A. C, shown is the mean and S.D. (error bars) for n = 14 replicates for protein expressed from chondrocytes harvested from different donors. The p value for one-way ANOVA comparing different H₂O₂ concentrations is <0.0001. Dunnett's multiple comparison test was used to identify H₂O₂ concentrations with significantly different JNK kinase activity from untreated protein; *, p value <0.05; ***, p value <0.005. D, JNK2 inhibition is reversible by reduction. Active, phosphorylated JNK2α2 was purchased from SignalChem and treated or not with H₂O₂ and then catalase as described above. Each sample was then split in half. DTT was added to one half (+), and an equal amount of water (−) was added to the second prior to the addition of c-Jun and ATP. p, phospho; t, total.