Mitochondrial localization of CSA and CSB in oxidatively stressed cells. Confocal laser scanning microscopy with green fluorescence for nuclear staining, blue fluorescence for mitochondrial staining, and red fluorescence staining for either CSA (A) or CSB (B). Pink fluorescence results from colocalization (white circles) of red mitochondrial CSA/CSB and blue mitochondrial staining. Pictures are representatives of at least five separate experiments. Bar, 1 µm. Exposure of cells to 25 µM H₂O₂ for 12 h leads to signal increase for mitochondrial CSA (C) and CSB (D). Histograms show signal intensities along the red line in the picture with simultaneous signal increment. (E) CSA-deficient fibroblasts transfected with CSA WT protein. Pictures are representatives of at least three independent experiments. Bar, 1 µm. (F) Whole-cell extracts (WCE) and mitochondrial extracts (Mt) prepared from H₂O₂-treated cells. Presence of mtDNA (IS) and absence of nuclear DNA (GAPDH), shown by PCR. Immunoblotting of mitochondrial ATP-synthetase β, phospho-NF-κB, S6, CSA, and CSB. Pictures are representatives of at least three independent experiments. (G) Electron microscopic image of oxidatively stressed normal human fibroblasts stained with gold-labeled (circles) CSA (top) or CSB (bottom). Bars: (top CSA image) 1.1 µm; (top CSB image) 1.6 µm; (bottom) 165 nm. Electron micrographs are representatives of at least two separate experiments. (H) Top: mt (10 µg protein per lane) isolated from H₂O₂-treated WT cells were incubated where indicated with 100 µg/ml proteinase K (PK) in the presence or absence of Triton X-100 (Tx-100). SDS-PAGE of mitochondrial proteins with antibodies against CSA, Tom20, Tim23, and superoxide dismutase (SOD). Bottom: mt (25 µg per lane) isolated from H₂O₂-treated WT cells were incubated with 25 µg/ml proteinase K (PK) in the presence or absence of Triton X-100 (Tx-100). SDS-PAGE of mitochondrial proteins with antibodies against CSB and cytochrome C. Pictures are representatives of at least two independent experiments.