FIGURE 2:

Lgd localization to endosomes is dependent on multiple factors. (A, C) Representative images of zygotes coexpressing a GFP fusion to Lgd and a mCherry fusion to STAM (A) or Rab5 (C) were acquired live using confocal microscopy (n = more than five animals each; zoomed portions of embryos are shown), in the presence and absence of various ESCRT proteins. Bar, 2 μm. (B, D) Quantification showing the relative level of overlap between Lgd and STAM (B) or Rab5 (D) under various depletion conditions as compared with control animals. **, p < 0.01 and *, p < 0.05, based on a t test (D) or an ANOVA followed by a Tukey post hoc test (B). (E) Purified recombinant Lgd was separated over a Wyatt gel filtration column coupled to a multiangle light-scattering system and fractions were separated by SDS–PAGE, followed by silver staining, to calculate its Stokes radius (top). UV absorbance (red) and light-scattering (green) profiles are also shown (bottom), which were used to calculate the molecular mass of Lgd (n = 3). (F) Recombinant Lgd (4 μM) was resuspended in a buffer containing Accudenz in the presence or absence of liposomes with two distinct lipid compositions and subjected to centrifugation. Protein–lipid complexes that floated to the surface were recovered by hand and subjected to SDS–PAGE, followed by silver staining (n = 3).