Fig. 3: Reducing E2 activity modulates several protein sets and organelle components.

A general, partial decline in E2 activity was obtained via UBA1 RNAi and via the concomitant RNAi-mediated knockdown of multiple E2s (siE2combo). a Western blot of HEK293T cells treated with siRNAs targeting UBA1 (siUBA1) versus control non-targeting siRNAs (siNT), with and without heat shock. b siUBA1 reduces the levels of detergent-insoluble ubiquitinated proteins. Heat shock (HS) increases the detergent-soluble levels of proteins modified with ubiquitin (total and K27, K48, and K63 linkage-specific ubiquitination) and with ubiquitin-like modifications (SUMO1 and SUMO2/3) but their levels are not modulated by siUBA1. The detergent-insoluble levels of ubiquitinated proteins (total and K48 linkage-specific ubiquitination, and SUMO1 modifications) decline in siUBA1- versus siNT-treated cells in the absence of heat shock; n = 3 biological replicates/group, mean ±SD, ***P < 0.001 (two-way ANOVA). c E2combo RNAi (a pool of siRNAs targeting 32 E2s) leads to a significant reduction in the mRNA levels of 19 E2s. The average of 3 biological replicates is shown, with red indicating a decline in E2 expression compared to control cells treated with the same amount of siNT. Bold-outlined boxes indicate a significant change (P < 0.05; unpaired two-tailed t test). d, e Detergent-soluble and insoluble ubiquitinated proteins decline in siE2combo-treated cells. Heat shock increases detergent-insoluble ubiquitinated proteins but this is hindered by E2combo siRNAs, especially for total ubiquitination and for K63 and SUMO1 modifications; n = 3 biological replicates/group, mean ±SD, ***P < 0.001 (two-way ANOVA). Proteomic and transcriptional changes induced by siUBA1 (f) and by siE2combo (h) versus siNT. Several proteins are upregulated and downregulated (P < 0.05; Log₂FC > 0.2 and <-0.2; red) without changes in the corresponding mRNAs. Proteins with corresponding mRNA changes induced by siUBA1 (P < 0.05; Log₂FC > 0.2 and <−0.2) are highlighted in blue and include siRNA-targeted UBA1 (f) and E2s (h), highlighted in violet. GO categories that are over-represented among proteins that are significantly (P < 0.05) upregulated (Log₂FC > 0.2) and downregulated (Log₂FC < −0.2) by siUBA1 (g) and by siE2combo (i) versus siNT, and that are not significantly modulated transcriptionally. Peroxisomal proteins are consistently upregulated in response to siUBA1 (f–g) and siE2combo (h, i). Source data are provided in the Source data file.