Figure 3.

Oxidation of DJ-1 affects complex formation. (A) Soluble complexes from SH-SY5Y cells and control human putamen brain samples were separated by BN PAGE using 7.5% BisTris gels and western blot analysis for DJ-1 was performed. (B) Left: Soluble protein complexes were extracted from SH-SY5Y, scrambled control (SC1), DJ-1 knock down (KD1 and KD2) cells and separated by BN PAGE on 7.5% BisTris gels. Middle: Transfection of WT DJ-1 into the KD background rescued complex formation after 72 h. Right: Over expression of WT DJ-1 in SH-SY5Y cells slightly increased the amount of complex after 72 h compared to untransfected SH-SY5Y cells. Representative western blots are shown (n > 3). (C) To compare total amounts of monomeric, dimeric and complex bound DJ-1, soluble protein complexes were extracted from SH-SY5Y cells, KD1 and KD1 cells transfected with WT DJ-1 or C106DD DJ-1 72 h after transfection. Complexes were separated on 8-16% gradient BisTris gels and western blot analysis was performed probing for DJ-1. Representative western blot results are shown (n > 3). Protein loading of samples used for BN-PAGE was assessed by measuring an aliquot of each sample for DJ-1 and β-actin level by SDS-PAGE (panels underneath BN PAGE). (D) Left: SDS PAGE and western blot analysis was performed with KD1 cells transiently transfected with wild type DJ-1 (WT), the oxidation deficient mutant DJ-1 form C106A (A) or the oxidation mimicking DJ-1 form C106DD (DD). Membranes were probed for oxidised DJ-1 (oxDJ-1) and total DJ-1. Right: SDS PAGE and western blot analysis was performed with KD1 cells, untransfected or transiently transfected with WT or DD DJ-1 Cells were either untreated or stress treated with 1 mM H₂O₂ for 1 h to induce oxidation of DJ-1. Membranes were probed for oxidised DJ-1 (oxDJ-1) and β-actin. (E) Soluble protein complexes were extracted from SH-SY5Y cells, KD1, KD1 expressing WT or C106DD DJ-1and separated by BN PAGE on 8-16% BisTris gels. Cells were either stress treated with 1 mM BSO for 24 h and subsequently 1 mM H₂O₂ for 30 min or left untreated. Left: short exposure probing for total DJ-1. Middle: longer exposure of the same membrane. Right: Membrane probed for oxidised DJ-1 (ox DJ-1). The dotted line denotes the molecular weight of WT DJ-1 complex under basal conditions and indicates that treatment induced oxidation of DJ-1 results in a slightly higher molecular weight of the complex (F) Soluble protein complexes were extracted from SH-SY5Y cells either stress treated (1 mM BSO for 24 h and subsequently 1 mM H₂O₂ for 30 min) or left untreated. Samples were separated by BN PAGE on 8-16% BisTris gels and western blot analysis for total and oxidised DJ-1 was performed. Left: longer exposure probing for total DJ-1. Middle: shorter exposure of the same membrane. Right: Membrane probed for oxidised DJ-1. The arrow indicates another complex detected by total DJ-1 antibody in oxidation treated SH-SY5Y cells with the same molecular weight as C106DD DJ-1 complex.