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. 2021 Mar 31;17(3):e1009463. doi: 10.1371/journal.pgen.1009463

Fig 1. Stx8 is required for the morphology and functionality of the prevacuolar endosome (PVE).

Fig 1

(A) Localization of Vps10-GFP in wild type (WT) and stx8Δ cells. Arrowheads denote cytoplasmic fluorescent dots. (B) The level of Vps10-GFP in cell extracts from the WT, stx8Δ and vps35Δ strains was analyzed by western blot. Cell extracts from a strain that did not express Vps10-GFP was used as a negative control. Tubulin was used as loading control. (C) Cpy1-Cherry distribution in the same strains as in (A). (D) Vps10-GFP distribution in the indicated strains. (E) WT cells bearing either GFP-Syb1 or Vps10-GFP, and stx8Δ cells bearing Vps10-GFP were treated with dimethyl sulfoxide (DMSO; solvent) and with latrunculin A for 20 minutes and photographed. (F) Distribution of the indicated PVE markers in WT and stx8Δ cells. (G) Distribution of the trans-Golgi network (TGN) marker Sec72-GFP in WT and stx8Δ cells. (H) Distribution of Ub:GFP-Cps1 in the indicated strains. For each image, the inset in the upper-right corner corresponds to an enlargement of the region delimited by dashed lines. (I) Equal amounts of protein in cell extracts from the indicated strains bearing Ub:GFP-Cps1 were subjected to western blotting with anti-GFP. The arrowhead denotes a 40 kDa band present in stx8Δ, and the arrow denotes the clipped GFP. The lower panel shows a low exposure of the gel region where the clipped GFP was detected. The experiment was performed three times. The lowest panel shows the mean, standard deviation, and statistical significance of the differences in the amount of GFP (a.u., arbitrary units), determined by Dunnett’s multiple comparisons tests after analysis of variance (ANOVA). ***, p < 0.001; ****, p < 0.0001. Images are single planes captured with a DeltaVision system. Scale bar, 5 μm.