Fig. 3: Mass spectrometry identifies endogenous substrates.

a Volcano plot shows significantly (q-value <0.1) increased (red) or decreased (blue) proteins in the dyf-1(mn335) mutant compared with WT derived by differential expression analysis of proteomic data. 3922 proteins were analysed in total, n = 3 biologically independent experiments. b Heat map of normalised protein intensity values (z-scores) derived by proteomic analysis of the indicated mutant strains. Proteins significantly (q-value <0.1, FDR corrected) increased in dyf-1(mn335) compared with WT and increased (p-value <0.05) compared with dyf-1(mn335) unc-13(e51) or dyf-1(mn335); unc-31(e928) double mutants are displayed, 2-sided, unpaired moderated t-test as calculated by limma package, n = 3 biologically independent experiments. Depicted schematic worms display the genotypes used and the expected levels of UbV-GFP substrate stabilisation. c Worms expressing the indicated mutations and ANMT-2::GFP were grown on control (−) or rpn-8(RNAi) (+) plates and analysed by Western blotting against GFP and tubulin, n = 4 biologically independent experiments. d Quantification of the experiments displayed in (c), n = 4 biologically independent experiments. WT versus rpn-8(RNAi) p = 0.04256; dyf-1(mn335) vs dyf-1(mn335) rpn-8(RNAi) p = 0.36806. 1-tailed unpaired Student’s t- test. e Worms expressing the indicated mutations and GFP::C05D11.5 were grown on control (−) or rpn-8(RNAi) (+) plates and analysed by Western blotting against GFP and tubulin, n = 4 biologically independent experiments. f Quantification of the experiments displayed in (e), n = 4 biologically independent experiments. WT versus rpn-8(RNAi) p = 0.0145; dyf-1(mn335) vs dyf-1(mn335) rpn-8(RNAi) p = 0.2621, 1-tailed unpaired Student’s t- test. d, f Column graphs: mean ± standard deviation, scatter plots, individual experiments, same colour: same experiment, *p ≤ 0.05, **p ≤ 0.01, n.s. p > 0.05. See also Supplementary Fig. 3 and 4 and Supplementary Data 1, Source data are provided as a Source Data file.