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. 2011 Sep 15;12(6):503–509. doi: 10.4161/cbt.12.6.15976

Figure 2.

Figure 2

HSP70 is required for ARF localization to mitochondria. (A) The HSP70 inhibitor phenylethynesulfonamide (PES) does not block HSP70-ARF complex formation. Co-immunoprecipitation of HSP70 with ARF antisera in U2OS-ARF cells following ARF induction with doxycycline for 24 h in the presence and absence of phenylethynesulfonamide (PES) at the indicated concentrations (5, 10, 20 uM). IgG is the negative control. IP: immunoprecipitation. (B) Co-immunoprecipitation of HSP70 with ARF from p53-/- MEFs following 24 h of treatment with 5 uM PE S. (C) Protein gel analysis using antisera for the proteins indicated of cytosolic (Cyto) and mitochondrial (Mito) extracts isolated from U2OS-ARF cells, treated or untreated for 24 h with doxycycline (Doxy, 100 ng/ml) and/or PE S (20 uM). The normalized values (norm. value) for ARF in the cytosol and mitochondria were assessed by densitometry and normalized to the BAK and PCNA controls. In the bottom part, 20 ug of mitochondrial and cytosolic extract were probed for GRP75 and BAK, which are mitochondrial proteins, and PCNA, which is cytosolic and nuclear, to assess the integrity of mitochondrial purification. (D) Immuno-electron microscopy using ARF antisera and Protein G-Gold was assessed for the percent of mitochondria with three or more gold labels in the mitochondria of U2OS-ARF cells following 24 h of treatment with doxycycline and/or PE S (20 uM). The values shown are averaged from three independent experiments. In the lower part, immuno-electron microscopy using ARF antisera followed by protein G-Gold is depicted in U2OS-ARF cells following 24 h of treatment with doxycycline and/or PE S (20 uM). The arrows indicate gold particles localizing with mitochondria. Scale bars are 500 nm. (E) Protein gel analysis using antisera for the proteins indicated from extracts isolated from whole cell lysate (WCL), cytosol (Cyto) and mitochondria (Mito) purified from mouse embryo fibroblasts (MEFs) from a wild-type (wt) or HSP70-/- mouse. In order to upregulate ARF, MEFs were infected for 48 h with short hairpin to silence p53 (shp53). Twenty ug of mitochondrial and cytosolic extract were probed for GRP75, which are mitochondrial proteins, and PCNA, which is cytosolic and nuclear, to assess the integrity of mitochondrial purification.