Fig. 4: Upregulation of peroxisomal proteins by UBA1/E2combo RNAi.

Modulation of the levels of peroxisomal proteins by UBA1 siRNAs (a) and E2combo siRNAs (b) compared to control NT siRNAs. The x-axis displays the Log₂FC of UBA1 RNAi (a) and E2combo RNAi (b) versus control NT RNAi whereas the y-axis reports the significance, indicated as -Log₁₀(P value; unpaired two-tailed t test). Overall, UBA1 and E2combo siRNAs upregulate the levels of several peroxisomal proteins, including peroxins (PEX). c, d PEX protein upregulation (P < 0.05 and Log₂FC > 0.2) upon E2combo knockdown occurs without significant and/or concordant mRNA changes. N = 3 biological replicates/group, mean ± SD (P values were obtained with unpaired two-tailed t tests for TMT data and with non-parametric ANOVA for RNA-seq data). e Expression of FLAG-tagged PEX proteins in HEK293T cells. UBA1 and E2combo RNAi increases the levels of PEX3-FLAG and PEX12-FLAG. N = 4 biological replicates/group with mean ± SD and P values indicated (*P < 0.05 and **P < 0.01; one-way ANOVA). f PEX turnover occurs at least in part via the proteasome. Immunoprecipitation of FLAG-tagged versions of some of the PEX proteins that are upregulated by UBA1/E2combo RNAi (a, b). Anti-ubiquitin (Ub) immunoblotting is used to determine the ubiquitination status of FLAG-tagged PEX proteins. Compared to controls (gray), treatment with the proteasome inhibitor MG132 (orange) increases PEX ubiquitination and this is largely prevented by concomitant treatment with the UBA1 inhibitor TAK243 (orange-green stripes). Similar results are obtained for PEX3, PEX5, PEX11A, PE11B, PEX12, and PEX13; n = 3 biological replicates/group, mean ± SD, *P < 0.05 (one-way ANOVA). These findings indicate that peroxin turnover occurs at least in part via the proteasome. See also Supplementary Fig. 9. Source data are provided in the Source data file.