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. Author manuscript; available in PMC: 2013 Jun 11.
Published in final edited form as: Nat Commun. 2012;3:1271. doi: 10.1038/ncomms2236

Fig. 6.

Fig. 6

Translocation of ErbB2 into mitochondria is kinase dependent. (A) The mitochondrial fractions and whole cell lysates were isolated and Western blotting was performed on MDA-MB-435ErbB2 (435eb), MDA-MB-435ErbB2V695E (435VE) and MDA-MB-435ErbB2K753M (435KM) cells. MtHSP70 and α-Tubulin were loading controls. (B) Mitochondrial fractions and whole cell lysates were isolated and Western Blotting was performed to detect mtErbB2 in mitochondria following the treatment of MCF7ErbB2 cells with ErbB2 inhibitor AG825 at 10 uM or control (DMSO) for 24 h; and with Heregulin β1 (HRG) at 10 ng/ml or control (PBS) for 24 h (upper). Whole cell lysates were subjected to Western Blotting to examine the phosphorylation status of ErbB2 and the total ErbB2. α-Tubulin was loading control (lower). (C) MCF7 cells were treated with and without HRG at 10 ng/ml for 24 h. The mitochondrial fraction and the whole cell lysate were isolated for Western blotting. Prohibitin and α-Tubulin were loading controls. (D) Left, mitochondrial proteins were isolated from SKBR3 cells and immunoprecipitated with anti-ErbB2 or anti-Cox II antibodies. The immunoprecipitates were analyzed by Western blotting with anti-ErbB2 and anti-Cox II antibodies. IgG was negative control. Right, mitochondrial proteins were isolated from 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells and immunoprecipited with Cox II antibody or negative control IgG. Samples were loaded on a SDS–PAGE followed by Western Blotting analysis with Cox II antibody to show the Cox II protein level, and with P-Y20 antibody to show the tyrosine phosphorylation level on Cox II. (E) Cytochrome c oxidase activities were measured after the cells were treated with or without a cytochrome c oxidase inhibitor Potassium cyanide (KCN) in 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells. The relative activities of Cox II were calculated relative to the Cox II activity of 231V cells. (F) 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells were treated with or without Taxol at 80 nM for 24 h. Cytosolic, mitochondrial fractions and whole cell lysates were analyzed by Western blotting with cytochrome c antibodies. α-Tubulin and mtHSP70 were cytosol and mitochondrial markers and loading controls. Columns, mean of three independent experiments; bars, SE.*, P < 0.05, **, P < 0.01