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. 2023 Nov 29;223(2):e202306107. doi: 10.1083/jcb.202306107

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© 2023 Li et al.

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Figure 2. — Animals lacking RABI-8 exhibit increased RAB-8 activity. (A) Confocal images of intestinal cells expressing GFP-tagged RAB-8. Compared with wild-type animals, the intensity of GFP::RAB-8–labeled puncta was increased in rabi-8(tm2518) mutants. (B) mCherry (MC)::RAB-8 coimmunoprecipitated by EHBP-1::GFP were increased in rabi-8 mutant animals. Levels of MC::RAB-8 are normalized to the corresponding GFP (set to 1 in wild-type animals). (C) The fluorescence intensity and the number of GFP::RAB-8 puncta were diminished in RABI-8–overexpressing animals. (D) Confocal images of intestinal cells expressing GFP-tagged PGP-1. Knockdown of SMGL-1 significantly reduced the cytosol accumulation of GFP::PGP-1 in rabi-8(tm2518) mutants. (E) Compared with wild-type animals, GFP::PGP-1 aberrantly accumulated in the cytoplasm of animals overexpressing RAB-8. For quantification, the signals from the apical membrane were avoided by manual ROI selection. Data are shown as mean ± SD (n = 18 each, six animals of each genotype sampled in three different unit regions of each intestine defined by a 100 × 100 [pixel²] box positioned at random). Statistical significance was determined using a two-tailed, unpaired Student’s t test. ***P < 0.001. Data distribution was assumed to be normal but this was not formally tested. (F and G) The localization of fluorescence-tagged ER maker SP12 (F) and Golgi marker AMAN-2 (G) was unaltered in rabi-8(tm2518) mutants. About one cell length of the intestine is shown in each panel. Scale bars: 10 μm. Yellow asterisks indicate intestinal lumen. Dashed lines indicate the outline of the intestine. Source data are available for this figure: SourceData F2.