CT behind PH domain mediates RTKN-1 self-association and RTKN-1 functions independently of PTRN-1 and SORB-1 in the intestine. (A) Western blot showing GST pulldown with in vitro–translated HA-tagged proteins. GST-RBD exhibited an interaction with HA-CT. (B) Native PAGE of uncrosslinked HA-RBD-PH and HA-RTKN-1. (C) GST-tagged proteins were separated on SDS-PAGE and stained with Coomassie blue. (D–E′) The actin-bundling capacity of HA-RTKN-1 was measured using the cosedimentation assay. α-Actinin was used as a positive control. Multiple concentrations were deployed to estimate the pellet/supernatant (P/S) ratio. Error bars are 95% CIs (n = 3 independent experiments). **, P < 0.01 by Student’s t test. (F–H′) Confocal images showing the subcellular localization of RTKN-1-GFP, EHBP-1-GFP, and GFP-Utrophin-CH in intestinal cells. Box-and-whisker plots (n = 18 cells): 10–90th percentile; dots, outliers; red midline, median of WT; boundaries, quartiles. **, P < 0.01; ***, P < 0.001 by Mann-Whitney test. (I–J′) The subcellular localization of RTKN-1-GFP or hTAC-GFP was not significantly affected in sorb-1(RNAi) cells. Box-and-whisker plots (n = 18 cells): 10–90th percentile; dots, outliers; red midline, median of WT; boundaries, quartiles. No significant difference by Mann-Whitney test. (K) mRNA level showing RNAi-mediated knockdown efficiency of SORB-1. Error bars are 95% CIs (n = 3 independent experiments). ***, P < 0.001 by Student’s t test. The black asterisks in the image panels indicate intestinal lumen. Scale bars, 10 µm. Ctrl, control; MC, mCherry.