FIGURE 3.

Western blot analysis of IscA1. A, cellular extracts containing either EGFP or EGFP-IscA1 were analyzed by Western blotting using anti-green fluorescent protein (GFP) (left) or anti-IscA1 (right) antibodies. Positions of molecular mass markers are shown to the left. B, HeLa cell cytosolic (40 μg) or mitochondrial (2 μg) extracts were analyzed by Western blotting using anti-IscA1 antibodies. Extracts (10 μg) were also analyzed by Western blotting using anti-β-tubulin or anti-cytochrome c (Cyto C) antibodies. C, whole cell extracts, mitochondrial matrix, mitochondrial membrane, and intermembrane space (IMS) fractions were analyzed by Western blotting using anti-IscA1, anti-HSP 60, or anti-cytochrome c antibodies. D, top panel, HeLa cell cytosolic extracts were incubated with or without the indicated antibodies, which were then immunoprecipitated (IP) with protein G-agarose. The immunoprecipitates or the extract input (representing 4% of the total) were then examined by Western blotting using anti-IscA1 antibodies. Bottom panel, HeLa cell cytosolic extracts were incubated with anti-IscA1 antibodies, which were then immunoprecipitated with protein G-agarose. The immunoprecipitates or the extract input (representing 4% of the total) before or after immunoprecipitation were then examined by Western blotting using anti-IscA1 antibodies. E and F, HeLa cells were transfected with control (CON) or IscA1 siRNAs. E, total RNA was isolated from cells and analyzed by real time PCR for IscA1 mRNA (means ± S.D.), and normalized to that of β-actin. F, mitochondrial extracts (20 μg) were analyzed by Western blotting using antibodies against IscA1 or cytochrome c.