Fig. 1: Defective sensory cilia mutants show reduced UPS-dependent protein degradation. | Nature Communications

Fig. 1: Defective sensory cilia mutants show reduced UPS-dependent protein degradation.

From: Thermosensation in Caenorhabditis elegans is linked to ubiquitin-dependent protein turnover via insulin and calcineurin signalling

Fig. 1

a Fluorescence and Nomarski images of worms expressing the UFD substrate in WT, dyf-1(mn335); dyf-1(+) and the direct sibling lacking the dyf-1 array (dyf-1(mn335)). DiI: ciliated amphid and phasmid neurons. Scale bar: 100 μm. b Protein lysates of worms displayed in (a) were analysed by Western blotting against GFP and tubulin. Dots: presence of indicated mutation. a, b n = 3 biologically independent experiments. c Protein lysates of indicated WT and mutant worms. n = 5 biologically independent experiments. d Protein lysates of wild-type, dyf-1(mn335) or hecd-1(tm2371) worms after ethanol (−) or 0.5 mg/mL cycloheximide in ethanol treatment for the indicated time points. Worms were analysed as in (b). long exp. = long exposure of the blot. e Quantification of 4 biologically independent experiments from (d), average ± standard deviation, one-way ANOVA with Bonferroni’s multiple comparisons test, hecd-1(tm2371) 1.5 h p = 0.0004, hecd-1(tm2371) 3 h p = 0.0012, dyf-1(mn335) 1.5 h p = 0.0166, dyf-1(mn335) 3 h p > 0.9999, ratios of mutants versus ratios of wild-type with cycloheximide. f WT and indicated mutant worms expressing the UFD substrate were analysed as in (b), but the top part of the blot exposed for an extended time to detect polyubiquitylated UbV-GFP, ivd-1(hh6): positive control to detect polyubiquitylated UbV-GFP86, n = 1. g WT or indicated mutant worms expressing UbV-GFP or the procathepsin L mutant CPL-1W32A,Y35A::YFP (CPL-1*-YFP), a marker for ER-associated protein degradation, are analysed by Western blotting against GFP and tubulin. h Quantification of the experiment displayed in (g) Column graphs: mean ± standard deviation, scatter plots: individual experiments, UbV-GFP: dyf-1(mn335) versus WT p = 0.0001, 2-tailed unpaired Student’s t- test, CPL-1*: dyf-1(mn335) versus WT p = 0.97, sel-1(e1948) versus WT p = 0.0013, One-way ANOVA followed by Dunnett’s multiple comparisons test, n = 3 (WT and dyf-1(mn335)) and n = 2 (sel-1(e1948)) biologically independent experiments. e, h ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05, n.s. p > 0.05. See also Supplementary Fig. 1. Source Data are provided as a Source Data file.

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