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Fig. 8. Overexpression of p50 alters Drp1 localisation to mitochondrial membranes. (A) Endogenous Drp1 was stained in (a) empty vector or (b) p50.EGFP-transfected cells (n=15 cells in three independent experiments). Boxed areas in are shown on an expanded scale below (c,d); image in right upper corner shows the distribution of mitochondria in the same cells. Notice the filamentous and dispersed staining of Drp1 in the images of control and in p50 cells. (B) Overexpression of wild-type Drp1 restores normal distribution of mitochondria in p50 cells. Mitochondria were visualised with MitoTrackerRed^TM in cells transfected with (a) empty vector, (b) p50.EGFP, (c) p50.EGFP+Drp1^wt and (d) Drp1^K38A (n=8 cells in three independent experiments). Hatched boundaries, obtained from an overlay with the transmitted image of the cell, indicate the cell periphery. Bars, 10 µm (C) Overexpression of p50 reduces the amount of Drp1 on mitochondrial membranes. Cells transfected with p50.EGFP (p50) or empty vector (Cont.) were fractionated as described in Materials and Methods. The post-nuclear supernatant (PNSN), post-mitochondrial supernatant (PMSN), mitochondrial pellet (Mito.P) and microsomal pellet (MP) were probed with a rabbit polyclonal anti-human Drp1 antibody. An equal amount of protein (26 µg/lane) was loaded from each fraction. The antibody recognised Drp1 at the expected size (80 kDa). (D) Overexpression of wild-type Drp1 restores the normal amount of Drp1 on mitochondrial membranes in p50-expressing cells. Post-nuclear supernatant (PNSN) and the mitochondrial pellet (Mito.P) were prepared from control cells (Cont.) and from cells overexpressing Drp1 (Drp1) and Drp1+p50 (Drp1+p50). An equal amount of protein (16 µg) was loaded in each lane. The blots were scanned and quantified with NIH ImageJ software (http://rsb.info.nih.gov/ij/).

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