Figure S4.
RTKN-1 interacts with F-actin but not with UNC-60A. (A and A′) The actin-binding potential of GST was measured 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). No significant difference by Student’s t test. (B) Coimmunoprecipitation assay analyzing RTKN-1–actin interaction. (C) GST-tagged UNC-60A and RTKN-1 were separated on SDS-PAGE and stained with Coomassie blue. (D) Western blot showing UNC-60A (18 kD) and UNC-60A-GFP (45 kD) in WT, unc-60a(RNAi), and UNC-60A-GFP animals. (E and E′) The endogenous level of UNC-60A or RTKN-1 (UNC-60A::2xFlag and RTKN-1::2xFlag CRISPR knockin strains). Relative intensity of UNC-60A or RTKN-1 was calculated with reference to the actin level, and the normalized level of RTKN-1 served as a standard (1.000). (F and F′) Confocal images showing colocalization between endogenous UNC-60A and PI(4,5)P2 reporter Tubby (Tb)-PH(R332H). The white arrowheads indicate structures labeled by both GFP and mCherry. Pearson's correlation coefficients were calculated; error bars are 95% CIs (n = 12 animals). (G and G′) The presence of GST-COR-1 or GST-POD-1 did not affect GST-UNC-60A–mediated actin depolymerization. GST-tagged proteins were separated on SDS-PAGE and stained with Coomassie blue. (H) Western blot showing GST pulldown with in vitro–translated HA-tagged UNC-60A. There was no interaction between RTKN-1 and UNC-60A. (I–J′) Confocal images showing RNAi-mediated knockdown of UNC-60A-GFP and the subcellular distribution of RTKN-1-GFP. 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. The black asterisks in the image panels indicate intestinal lumen. Scale bars, 10 µm. Ctrl, control; IB, immunoblot; IP, immunoprecipitation; P, pellet; S, supernatant.

RTKN-1 interacts with F-actin but not with UNC-60A. (A and A′) The actin-binding potential of GST was measured 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). No significant difference by Student’s t test. (B) Coimmunoprecipitation assay analyzing RTKN-1–actin interaction. (C) GST-tagged UNC-60A and RTKN-1 were separated on SDS-PAGE and stained with Coomassie blue. (D) Western blot showing UNC-60A (18 kD) and UNC-60A-GFP (45 kD) in WT, unc-60a(RNAi), and UNC-60A-GFP animals. (E and E′) The endogenous level of UNC-60A or RTKN-1 (UNC-60A::2xFlag and RTKN-1::2xFlag CRISPR knockin strains). Relative intensity of UNC-60A or RTKN-1 was calculated with reference to the actin level, and the normalized level of RTKN-1 served as a standard (1.000). (F and F′) Confocal images showing colocalization between endogenous UNC-60A and PI(4,5)P2 reporter Tubby (Tb)-PH(R332H). The white arrowheads indicate structures labeled by both GFP and mCherry. Pearson's correlation coefficients were calculated; error bars are 95% CIs (n = 12 animals). (G and G′) The presence of GST-COR-1 or GST-POD-1 did not affect GST-UNC-60A–mediated actin depolymerization. GST-tagged proteins were separated on SDS-PAGE and stained with Coomassie blue. (H) Western blot showing GST pulldown with in vitro–translated HA-tagged UNC-60A. There was no interaction between RTKN-1 and UNC-60A. (I–J′) Confocal images showing RNAi-mediated knockdown of UNC-60A-GFP and the subcellular distribution of RTKN-1-GFP. 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. The black asterisks in the image panels indicate intestinal lumen. Scale bars, 10 µm. Ctrl, control; IB, immunoblot; IP, immunoprecipitation; P, pellet; S, supernatant.

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