LST-6/DENND5 acts as a GEF for RAB-10. (A and A′) Confocal images showing the distribution of VIT-2-mNeonGreen-3xFlag^KI in the C. elegans intestine, oocytes, and embryos in different gene knockdown backgrounds. (B and B′) Confocal images showing the subcellular localization of mNeonGreen-3xFlag-RAB-10^KI. The statistical data are shown as box-and-whisker plots with 10th–90th percentile (n = 24 cells from eight animals of each genotype; dots, outliers; boundaries, quartiles; one-way ANOVA test with Dunn’s multiple comparison in A′; two-tailed Mann-Whitney test in B′). (C and C′) Western blot analysis of the membrane-to-cytosol ratio of RAB-10 in control or lst-6(RNAi) animals. Band intensity was quantified by using the “Plot Lanes” function in Image J; error bars are 95% CIs (two-tailed Student’s t test) (n = 3 independent experiments). S: supernatant; P: pellet. (D and E) Western blotting showing GST pull-down detected protein interactions. (F) The rate constant of GTP exchange was analyzed using nonlinear regression (Exponential-One phase decay, Y0 = 100, Plateau = 50). A scatter diagram with protein concentration as the x-axis and GEF rate constant as the y-axis was generated and analyzed using linear regression. The slope and the slope significantly non-zero P value were shown. (G and G′) Confocal images showing colocalization between VIT-2-mNeonGreen-3xFlag^KI and mitochondria. The statistical analysis of colocalization was calculated as Pearson’s correlation coefficient and shown as mean ± SD (n = 18 cells from six animals of each genotype; two-tailed Mann–Whitney test). (H–H″) Western blot analysis of apoB protein levels in Huh7 cells. Band intensity was measured using the “Plot Lanes” function in ImageJ from three independent experiments. Error bars represent 95% CIs (one-way ANOVA test with Dunn’s multiple comparison). Source data are available for this figure: SourceData F7.