Skip to main content
. 2020 Jun 23;9:e53889. doi: 10.7554/eLife.53889

Figure 3. The Marie Kondo E2 conjugating enzyme mediates the degradation of ME31B, TRAL, and Cup.

Figure 3.

(A) Marie Kondo (Kdo/UBC-E2H) is required for the degradation of ME31B-GFP. Embryos laid from female flies expressing the indicated dsRNAs and ME31B-GFP were imaged at various time points by fluorescence microscopy. Fluorescent images are false-colored so that more intense fluorescence is indicated by hotter colors; fluorescence scale is shown. (B) Kdo is depleted in knockdown lines. An antibody was raised against Kdo, and 1–2 hr embryo lysates with the indicated dsRNA were probed for Kdo and PABP (as a loading control). (C) Kdo is required for destruction of ME31B, TRAL, and Cup. Staged embryos with mCherry or Kdo knocked down were harvested at the indicated times. Western blotting was performed on the lysates, probing for ME31B, Cup, TRAL, and eIF4E (as a loading control). Because the maternal flies contained wild-type and ME31B-GFP alleles, both proteins are visible in the α-ME31B blot. (D) Kdo is required for the ubiquitination of ME31B. ME31B-GFP was immunoprecipitated from 1 to 2 hr mCherry or Kdo knock-down embryo lysates under stringent conditions and then analyzed by western blotting by probing with α-GFP, α-eIF4E, or α-ubiquitin. IgG was used as an immunoprecipitation control.