<p>(A, B) Whole cell lysates were prepared from control mouse ear fibroblasts or liver) and immunoprecipitated (IP) with antibodies to Drp1, Mfn2 and OPA1 as indicated. For each blot, w1 corresponds to input, and w2, w3 and w7 correspond to wash number. IP is the immunoprecipitate, and the position of AnxA6 is indicated. Blots were probed with antisera to Drp1 (A) and AnxA6 (B), the position of which is indicated, and visualised using enhanced chemiluminescence. The band running beneath AnxA6 in the Mfn2 and OPA-1 IP lanes is IgG heavy chain. (C) Purified mitochondria and whole cell lysates of liver and primary fibroblasts from control (Wt) and <i>AnxA6</i><sup>−/−</sup> (KO) mice were immunoblotted with antisera against Drp1 and the mitochondrial marker Tim23 as a control for loading. (D) Fibroblasts from control and <i>AnxA6</i><sup>−/−</sup> mice were immunostained with antisera to Drp1 (red) and the mitochondrial marker cytochrome C (green), and analysed by confocal microscopy. The insets show reticular staining of Cyto C in control fibroblasts, with little co-localisation with Drp1 (visualised in orange), in contrast to increased co-localisation of the two proteins in the AnxA6 null cells. Scale bar = 5 µm. (E) The proportion of Drp1 immunofluorescence coincident with Cyto-c was calculated using Metamorph, and is presented as mean ± s.d., n = 3000 cells in 3 separate experiments, *p<0.05. (F) A431 cells stably expressing AnxA6 were triple stained for AnxA6 (green), Drp1 (red) and Cytochrome c (magenta). Regions that appear white in the lower right zoomed panel indicate coincidence of the three antigens. Scale bar = 2 µm. (G) A431 cells stably expressing AnxA6 were simulated with 1 µm ionomycin for 5 min, then fixed and stained for AnxA6, Drp1 and Cytochrome c as in (F). Note that AnxA6 relocates from the cytosol to plasma membrane in cells exposed to ionomycin (lower right ‘merge’ panel), with loss of regions of coincident staining of the three proteins (seen as white in the top right ‘merge’ panel). Scale bar = 5 µm.</p
