Figure 2. ME31B is degraded by the ubiquitin-proteasome pathway.

(A) Ubiquitination of ME31B-GFP requires the PNG kinase. ME31B-GFP was immunoprecipitated under stringent conditions from 1–2 hr lysates from wild-type or png⁵⁰ embryos, and then analyzed by western blotting by probing with α-GFP, α-eIF4E or α-ubiquitin. (B) ME31B-GFP is not degraded by autophagy. Embryos 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. (C) Inhibiting the proteasome partially stabilizes ME31B-GFP. As in B, except for dsRNAs targeting mCherry or Rpn10, a proteasome component. (D) ME31B-GFP is stabilized in Rpn10-depleted embryos. ME31B-GFP fluorescence from (C) and intervening time points was quantified. For each embryo, the fluorescence was normalized to its intensity at 0 min, and the fraction remaining is plotted through the time course. Significance was calculated using the Mann-Whitney test. (E) Depleting the proteasome partially stabilizes ME31B-GFP. Staged embryos from the indicated times were harvested with mCherry or Rpn11 knocked down. Western blotting was performed on the lysates, probing for GFP and eIF4E (as a loading control). Related to Source Figure 2—source data 1.