Figure 2. Perturbations of PINK1 and parkin specifically influence dMfn abundance.

Protein extracts from wt males (m), PINK1^B9 hemizygous males, wt males and females (m and f) and park²⁵ males and females were subjected to western blot analysis with an affinity-purified anti-dMfn antiserum (A), an anti-Opa1 antiserum (C), an affinity-purified anti-Drp1 antiserum (D), an anti-complex V β antiserum (A, C, D), an anti-VDAC antiserum (A, C, D), and an anti-actin antiserum (A, C, D). Arrow in (A) indicates location of the dMfn band at 94 kDa and the asterisks (*) indicate nonspecific bands. (B) Quantification of dMfn abundance in PINK^B9 and park²⁵ null mutants relative to wt controls. Because PINK1^B9 males are sterile and the PINK1 gene resides on the X chromosome, we are only able to generate male PINK1^B9 mutants and thus used wt males as the control population for these mutants. The ratio of dMfn to actin abundance was obtained from three independent blots for each sample analyzed; the ratios for wt controls were set at a value of 1 and mutant ratios were normalized to the wt ratios. *p<0.05 by Student’s t-test. (E) Protein extracts from mitochondrial and cytosolic fractions (see Materials and Methods section for details on fractionation) were subjected to western blot analysis with an affinity-purified anti-Drp1 antiserum, an anti-complex V β (CompV) antiserum, an anti-VDAC antiserum, and an anti-actin antiserum. (F) Protein extracts from wt flies, flies overexpressing Parkin (hsp70-GAL4/UAS-Parkin) and flies overexpressing PINK1 (hsp70-GAL4/UAS-PINK1) were subjected to western blot analysis with an affinity-purified anti-dMfn antiserum, an anti-complex V β antiserum, an anti-VDAC antiserum, and an anti-actin antiserum. Flies were subjected to a 1-hr heat shock and collected for analysis 24 hrs following the heat shock. The arrow indicates the location of the dMfn band at 94 kDa and the asterisks (*) indicate the two nonspecific bands.