Fig. 3: UFD1s modulates K63-polyubiquitination of UFD1f via competitively binding to MARCH7.
Ubiquitination of UFD1f examined by western blotting with α-total, K48-, K11-, K63-, and K6-linked polyubiquitin antibodies in WT or MUT cells. HEK293T cells (a) and N2a cells (b). (Ub)n, polyubiquitin. c, d Total, K48-, K11-, K63-, and K6-linked ubiquitination of UFD1f with or without UFD1s overexpression. HEK293T cells (c) and N2a cells (d). e E3 ubiquitin ligases co-immunoprecipitated by UFD1s and UFD1f. IP-MS immunoprecipitation followed by mass spectrometry. f Interactions between HA-tagged MARCH7 and UFD1f in HEK293T and N2a cells upon UFD1s deficiency (MUT) or overexpression (OE), examined by co-IP. g Total and K63-linked ubiquitination of UFD1f in HEK293T and N2a cells upon MARCH7 knockdown. ShCOO2, shRNA control with scrambled sequences. h Experimental procedure for a two-step IP assay (Re-IP) followed by MS to identify K63-ubiquitinated sites of UFD1f in HEK293T cells. i K63-ubiquitination detection of overexpressed Flag-tagged UFD1f in HEK293T cells. Flag-tagged wild-type, UFD1fK240R, or UFD1fK240R/K280R UFD1f was overexpressed. HA-K63Ub, a plasmid expressing HA-tagged ubiquitin that can only form K63-linked ubiquitination. All quantifications (Quan.) are normalized to the immunoprecipitated UFD1f, and quantitative 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.
