Fig. 2.

Oxidation of DJ-1 is prevented in the C106A mutant. (a) Endogenous DJ-1 in M17 cells responds to oxidative stress. (Upper) From left to right, blots of 2D gels from extracts from untreated cells, cells exposed to PQ²⁺ or MPP⁺; on the right, from cells exposed to PQ²⁺ for 24 h then placed in fresh media for 24 h. Blots were probed with monoclonal anti-DJ1 (arrow), which shows multiple isoforms: numbers below the blots show approximate pI values for each of the major isoforms. (Inset) Area around the pI 5.8 isoform, which accumulates after oxidative stress. (b) Response to oxidative stress in cysteine mutants. Samples were extracted from cells transfected with V5-tagged WT, C53A, or C106A DJ-1, from either untreated cells or cells exposed to PQ²⁺. Blots were probed with monoclonal anti-V5 and the pI 5.8 isoform identified (indicated below). The pI 5.8 isoform accumulated with both WT and C53A variants but not with the C106A variant (Bottom). Quantification of this and three similar experiments in c shows the ratio of pI 5.8:pI 6.1 isoform (n = 4; bars represent SEM). Note that the y axis scales are different for C106A; we included the faint immunoreactivity in the pI 5.8 region, but the ratio was still substantially lower than for the other variants.