Fig. 5: Linear ubiquitination of SMURF1 attenuates its E3 activity. | Nature Communications

Fig. 5: Linear ubiquitination of SMURF1 attenuates its E3 activity.

From: Systematic HOIP interactome profiling reveals critical roles of linear ubiquitination in tissue homeostasis

Fig. 5

a Immunoprecipitation of ectopic expressed HOIP protein in nine tissues lysates and immunoblot with the SMURF1 antibody. Immunoprecipitates of SMURF1 in 3T3-E1 cells (b) and immunoprecipitates of HOIP in BMSC cells (c), followed by immunoblotting with the indicated antibodies. d Ni-NTA pull-down analysis of ILK linear ubiquitination in HEK293T cells transfected with LUBAC and immunoblot with the indicated antibodies. e Immunoprecipitates of SMURF1 linear ubiquitination in HEK293T cells transfected with LUBAC wildtype and inactive mutant. f Immunoprecipitates of endogenous SMURF1 ubiquitination in HEK293T cells transfected with LUBAC. g Immunoblot of SMURF1 and its substrate SMAD1/5 in HOIP knockdown BMSCs. h Immunoblot of SMURF1 and its substrate SMAD1/5, MEKK2 in BMSCs transfected with Flag-tagged HOIP. i Immunoblot analysis of the interaction between SMURF1 and its E2 UBCH5C, UBC12 in vitro. j Three-dimensional structure of the SMURF HECT domain (PDB: 1ZVD). Linear ubiquitin lysine sites in blue and catalytic center in red. k Immunoprecipitates of SMURF1 and its KR mutants’ linear ubiquitination in HEK293T cells transfected with LUBAC. l Immunoprecipitates of SMAD1 ubiquitination in DMSO or HOIPin-8 treated HEK293T cells transfected with wildtype SMURF1, SMURF1 KR mutant or wildtype LUBAC. Source data are provided as a Source Data file.

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