Fig. 7: Post-translational regulation of VHP-1 stability by components of the ubiquitin-proteasome system.
From: Trauma-induced regulation of VHP-1 modulates the cellular response to mechanical stress
a Western blot time-course of VHP-1::GFP following treatment with DMSO (vehicle) or 50 µM MG132. Tubulin, load control. b Western blot of VHP-1::GFP without injury (control, C) or 1 and 4 hours (h) post injury. Worms treated with ubq-1 RNAi for 24 h. #ubq-1 RNAi sensitive high-MW bands. c Co-immunoprecipitation of VHP-1::GFP immunoblotted with anti-ubiquitin. Uninjured and injured non-transgenic worms (N2) as control. Input, VHP-1::GFP and tubulin from total worm extracts. d Effects of math-33 (deubiquitinase, squares) or vhp-1 (triangles) RNAi on transgenic worms expressing VHP-1::mCherry in all tissues (PMD117). Flow cytometry data depicts the proportion of worms falling into a high-fluorescence gate. Data are proportional to EV control from each independent experiment. Mean ± SEM, ****p < 0.0001 by one-way ANOVA with Sidak test. e Western blot of VHP-1::GFP from worms before (C, control) or 1 and 4 hours (h) after injury treated with math-33 RNAi. Tubulin, loading control. n = 4, uninjured and 1 h post injury, n = 3, 4 h post injury. f Quantification of western blots from Fig. 7e. Mean ± SEM. **p = 0.0088, *p = 0.0427, not significant (ns) p = 0.8134 by two-way ANOVA with Tukey test. g Co-immunoprecipitation of VHP-1::GFP (anti-GFP IP) from injured worms on empty vector (EV) or math-33 RNAi immunoblotted with anti-ubiquitin. Injured, non-transgenic N2 worms as control. Input, VHP-1::GFP, and tubulin from total worm extracts. n = 4 biological replicates. h qPCR showing relative math-33 transcript abundance in wild type and kgb-1(um3) mutant worms with (Inj) and without (Uninj) injury. Mean ± SEM, *p = 0.0135 by two-way ANOVA with Sidak test, WT n = 6, kgb-1(um3) n = 3. i Large-particle flow cytometry of wild type, PMD13, and math-33(ok2974) mutants, PMD173, two days post trauma. Dopaminergic GFP index indicates relative loss of dat-1p::GFP signal of injured math-33(ok2974) mutants compared to injured wild type worms. Mean ± 95% CI. ****p < 0.0001 by two-sided paired t-test. n = 4149, wild type, and n = 2601, math-33(ok2974), over three independent trials. Positive values indicate reduced loss of fluorescence when compared to age-matched uninjured controls. A value of one indicates no change of fluorescence compared to uninjured controls.
