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. 2005 Dec 2;7(3):341–345. doi: 10.1038/sj.embor.7400601

Figure 1.

Figure 1

Alternative oxidase is expressible in human cells and targeted to mitochondria. (A) Simplified biochemical scheme of mitochondrial respiration and by-pass of the cytochrome segment provided by alternative oxidase (AOX). The five complexes of the respiratory chain (RC) are denoted by Roman numerals. (B) Immunoblot of 20 μg total cell lysate from Flp-In™ T-REx™-293 (Invitrogen) cell clones transfected either with AOX–Flag or AOX–Myc constructs, or empty vector, and probed with primary antibodies is shown. Lanes denoted (+) were lysates from cells treated with 1 μg/ml doxycyclin to induce transgene expression. Primary antibodies used were mouse anti-Myc monoclonal 9E10 (anti-Myc) and anti-Flag M2 antibody (anti-Flag). (C–E) Fluorescence micrographs of cells transfected with the AOX–Flag construct. (C) Immunocytochemistry using anti-Flag M2 primary antibody. (D) Staining with Mitotracker® Red (Molecular Probes). (E) Superposition of the images from (C,D). Immunocytochemistry was carried out as described by Garrido et al (2003).