RTKN-1 acts independently of the Rho family of GTPases during recycling. (A) Western blot showing GST pulldown with in vitro–translated HA-tagged RTKN-1. RTKN-1 showed an interaction with RHO-1 loaded with GMP-PNP. RTKN-1 displayed no binding to CDC-42 loaded with GDP or GMP-PNP. Also, RTKN-1 displayed no binding to CED-10 loaded with GDP or GMP-PNP. (B–E′) Western blot and confocal images showing RNAi-mediated knockdown efficiency of GFP-RHO-1, GFP-CDC-42, and GFP-CED-10. Box-and-whisker plots (n = 18 cells): 10–90th percentile; dots, outliers; red midline, median of WT; boundaries, quartiles. ***, P < 0.001 by Mann-Whitney test. (F–H′) Confocal images showing subcellular localization of RTKN-1-GFP, hTAC-GFP, and hTfR-GFP in intestinal cells. Box-and-whisker plots (n = 18 cells): 10–90th percentile; dots, outliers; red midline, median of WT; boundaries, quartiles. ***, P < 0.001 by one-way ANOVA followed by Dunn’s post hoc multiple comparison test. Scale bars, 10 µm. The black asterisks in the image panels indicate intestinal lumen. Ctrl, control; MC, mCherry.