Identification of abnormalities of respiratory complex subunits in DJ-1-deficient cells.

<p><b>(A-a):</b> SN4741 represents dopaminergic neuronal cells and DJ-1(−/−) represents dopaminergic neuronal cells homozygous for a DJ-1 gene deletion. To compare the native state of the respiratory complexes in these cell lines, isolated mitochondria were solubilized by n-dodecyl-β-d-maltoside (DDM) and resolved by using blue native-polyacrylamide gel electrophoresis (BN-PAGE). After transferring to a nitrocellulose membrane, the blot was incubated with monoclonal antibodies against five mitochondrial complex subunits using MS601 antibody cocktail (MitoSciences, Eugene, OR). The molecular weight of mitochondrial complex I was about 1000 kDa. Normalization of isolated mitochondria was achieved based on HSP60 protein expression level. The result shown is representative of three independent experiments. <b>(A-b):</b> Protein expression levels were analyzed quantitatively by densitometry. Error bars represent the mean ± SD. Significant difference compared to SN4741 cells: ***, p<0.001. <b>(B):</b> Isolated mitochondria solubilized by digitonin were analyzed by BN-PAGE for supercomplex detection. Supercomplex was detected by using MS601 monoclonal antibody cocktail as described above. I+III₂+IV: supercomplex formed of complex I, dimeric complex III, and complex IV. I+III₂: supercomplex formed of complex I and dimeric complex III. III₂+IV: supercomplex formed of dimeric complex III and complex IV. III₂: supercomplex formed of dimeric complex III <b>(C-a):</b> 2D gel analysis. BN-PAGE was performed with mitochondria isolated from SN4741 cells or DJ-1 null cells to obtain the first dimension of resolved native complexes. By using BN-PAGE gels strips, further separation in the second dimension was performed by SDS-PAGE. In DJ- null cells, in the complex I lane of the 2D gel, the uppermost dot is missing; compare the circles marked by asterisks in the two panels. <b>(C-b,c):</b> Quantification of complex I ‘dot’ levels. Two dots present in SN4741 cells were compared to the dots of DJ-1 null cells in the same position. The upper dot levels in each cell line were normalized to the lowest dot in the complex I lane of the respective cell lines and the value obtained for the DJ-1 null cells was compared to that of the SN4741 cells; the results are derived from three independent experiments. <b>(D-a, b):</b> The spot on the gel was analyzed by mass spectroscopy and determined to be the complex I subunit NDUFS1.