Figure 1.

Strap is localised at the mitochondria and interacts with ATP synthase. (a) Endogenous Strap from various cell types was detected in total lysate (TL), mitochondrial (mit) and cytoplasmic (cyt) fractions. Cytochrome oxidase IV (COXIV) was used as a mitochondrial marker. PCNA was used as a control for nuclear contamination. The mitochondrial fractions were estimated to be 95% enriched. (b) U2OS cells were transfected with non-targeting (NT) or Strap (S) siRNA for 72 h. Mitochondrial fractions were prepared and immunoblotted with the indicated antibodies. COXIV served as a mitochondrial marker. (c) Endogenous Strap from U2OS cells was detected in mitochondrial (mit) fractions, prepared using different extraction techniques (1 and 2; see Materials and Methods). Nuclear and endoplasmic reticulum material was monitored using PCNA and calnexin antibodies respectively. COXIV served as a mitochondrial marker. Total cell lysate (tot) serves as a comparison. (d) U2OS cells were transfected with L-Strap and immunostained with an anti-HA antibody to detect ectopic Strap. DAPI was used to visualise the nuclei and cytochrome c for mitochondria. The merged imaged is shown (magnification × 600). (e) L-Strap (L) or control (-) transfected U2OS cells (i) were immunoprecipitated (IP) with anti-Flag antibody and the indicated silver-stained protein bands (ii) excised and subjected to tryptic digestion and liquid chromatography tandem mass spectrometry; the position of L-Strap and ATP synthase β-subunit is indicated