(A) Representative fluorescence images of tetracycline-induced HEK293T cells that contained the Control empty vector (pCDNA5-FRT/TO) or hTim10b3XFLAG. Cells were fixed prior to incubation with anti-FLAG (left panel, green; to stain hTim10b3XFLAG) and anti-NDUFAF2 (middle panel, red; to visualise the mitochondria). Hoechst stain (far right panel, blue) was used to stain the nucleus. Primary antibodies were counterstained with Alexa Fluor 568 and 488 secondary antibodies prior to microscopy. Scale bar: 10 µm. (B) Mitochondria isolated from control cells or cells expressing hTim10b3XFLAG were solubilised in digitonin-containing buffer before being analysed by BN-PAGE and immunoblotting using anti-FLAG antibodies. (C) [35S]-labelled hTim10b precursor was imported into mitochondria isolated from wild type HeLa cells for the indicated times in the presence or absence of a membrane potential (Δψ). Following import the mitochondria were re-isolated, lysed in digitonin-containing buffer and subjected to blue native electrophoresis and autoradiography. Asterisk (*) indicates non-specific band. (D) Mitochondria isolated from control and hTim10b expressing cells were solubilised in digitonin-containing buffer. Mitochondrial lysates were subjected to immunoprecipitation with anti-FLAG resin. Collected fractions were analysed by SDS-PAGE and western blotting using anti-FLAG and anti-SDHA antibodies. T, Total; P, Pellet; S, Supernatant; UB, Unbound; WI and WII, Wash I and II and E, Elution. 5% of the T, P, S, UB, WI and WII fractions and 100% of the E fraction were loaded for SDS-PAGE analysis. (E) Volcano plot showing proteins enriched in hTim10b pull-down versus the empty vector control. All proteins were plotted and each circle represents one protein/gene. The X-axis shows the Log2 fold change of Tim10b interacting partners and Y-axis shows the −log10 of the p-values.