N-terminal RBD and PH-CT are required for RTKN-1 functionality. (A) Domain architecture of RTKN-1a and fragments. (B and B′) The actin-binding potential of the purified GST-RBD or GST-PH-CT was measured by using the cosedimentation assay. Band intensity was quantified by using the “Plot Lanes” function of ImageJ; error bars are 95% CIs (n = 3 independent experiments). ***, P < 0.001 by Student’s t test. (C and C′) Confocal images showing subcellular localization of RTKN-1-GFP, RBD-GFP, and PH-CT-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. (D–F′) Confocal images showing the distribution of GFP-Utrophin-CH, 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. The black asterisks in the image panels indicate intestinal lumen. Scale bars, 10 µm. Ctrl, control; MC, mCherry; P, pellet; S, supernatant.