Fig. 4: UFD1s regulates ubiquitination dynamics of proteins, including IPMK and IPMK is regulated by UFD1f and MARCH7.

a Schematic workflow of ubiquitin-modified proteome. Briefly, ubiquitin tags of total proteins were digested to yield di-glycine (GG) linked to K, and then K-GG-modified peptides were subjected to IP-MS (see “Method” for details). b Volcano plots illustrating changes of ubiquitination levels of individual proteins in MUT versus WT HEK293T cells. Red or blue dots represent proteins with statistically significantly up or downregulated ubiquitination levels. IPMK has the most increased ubiquitination levels in MUT as compared to WT cells. c GO analysis of 178 proteins with significantly upregulated ubiquitin levels in MUT HEK239T cells. d Western blotting revealing IPMK protein levels in WT and MUT HEK293T or N2a cells treated with or without 20 μg/mL cycloheximide (CHX) for 4 h. Total, K48-, K11-, and K63-linked ubiquitination levels of IPMK in WT and MUT HEK293T (e) or N2a (f) cells. g UFD1f IP could co-IP IPMK in HEK293T cells. h IPMK protein levels in HEK293T cells expressing Flag-tagged UFD1f WT or K240R/K280R double mutant. i K48- and K11-polyubiquitination levels of IPMK in HEK293T cells under the overexpression of Flag-tagged UFD1f WT or K240R/K280R double mutant. j Western blotting revealing IPMK protein levels in HEK293T and Hepa1-6 cells after MARCH7 knockdown. k K48- and K11-polyubiquitination levels of IPMK in HEK293T and Hepa1-6 cells upon MARCH7 knockdown. ShCOO2, shRNA control with scrambled sequences. For (d, h, j), quantification was normalized to ACTIN levels; for (e, f, i, k), quantification was normalized to the immunoprecipitated IPMK levels. Data from three independent experiments are shown as mean ± SEM. P values were calculated by two-tailed Student′s t-test. Source data are provided as a Source Data file.