Extended Data Fig. 1: Miro Binds to dMIC60 in a Redox-Dependent Manner.
(a) Two-day-old flies expressing Myc-tagged UAS-dMIC60-WT or dMIC60-CS driven by Actin-GAL4 in dMIC60 null background13,28, were lysed and immunoblotted. Both anti-dMIC60 and anti-Myc recognize transgenic dMIC60 protein in dMIC60 null background (no endogenous dMIC60)28. Anti-dMIC60 recognizes endogenous dMIC60 in flies with Actin-GAL4 alone in wild-type background. (b) Whole-body lysates of wild-type flies (w1118) were IPed with anti-DMiro or IgG, and immunoblotted (IB) as indicated. (c) Immunostaining of dMIC60 and ATP5β in third instar larval muscles. Scale bar: 5 μm. (d) Immunostaining of T7 in adult fly brains (day 7). Scale bar: 20 μm. (c-d) Confocal images were obtained using the same settings. (e) In Vitro GST pull-down using full-length GST-Miro1 or GST, together with recombinant dMIC60 (AAs 92-739). (f) In Vitro GST pull-down using full-length GST-Miro1, together with recombinant dMIC60 (AAs 92-739), either wild-type (WT) or mutant (CS). (g) Coomassie-stained gels. Arrowhead indicates the dMIC60 band. (h-i) In Vitro GST pull-down using full-length GST-Miro1 or GST, together with recombinant dMIC60 (AAs 92-739) (h) or Parkin (i). (a-i) Similar results were seen more than three times. (j) Lysates of wild-type flies fed with H2O2, paraquat, or vehicle (water) at day 7 for 24 hrs were immunoblotted as indicated. The band intensity of each marker is normalized to that of β-actin from the same blot and graphed as relative change compared to vehicle. n=4 independent experiments. Two-sided Student T Test. p=0.0336 (DMiro, H2O2), 0.0325 (dMIC60, H2O2), 0.0176 (DMiro, paraquat), 0.0383 (dMIC60, paraquat). Boxes show 25th/75th percentiles, whiskers are the minimum and maximum, and middle line is median.
