Fig. 3: OTULIN inhibits linear ubiquitination of β-catenin.

a M1-linked ubiquitination of immunoprecipitated β-catenin from MDA-MB-231 cells transfected with LUBAC (HOIP + HOIL1) or along with HA-OTULIN. b UbiCREST analysis of β-catenin ubiquitination. MDA-MB-231 cells were treated with MG132 (10 µM) for 16 h. Then, β-Catenin was immunoprecipitated and incubated with indicated DUBs. The ubiquitination of β-Catenin precipitates after DUB digestion was analyzed by Western blot. c Linear ubiquitination of immunoprecipitated β-catenin from parental or HOIP knockout HEK293T cells. d Immunoblotting of co-immunoprecipitated proteins with β-Catenin in MDA-MB-231 cells treated with Dox (2 µg/ml) and MG132 (10 µM) for 8 h. e Linear ubiquitination of β-Catenin immunoprecipitates from parental and OTULIN knockout MDA-MB-231 cells treated as indicated. f Levels of linear Ub chain and K48 Ub chain in β-Catenin immunoprecipitates from parental, OTULIN knockout MDA-MB-231 cells or OTULIN-KO cells reconstituted with OTULIN-WT or –C129S mutant. *: endogenous OTULIN. g Linear and K48 ubiquitination of Flag-β-catenin WT, K133R, K19R, K49R, or K19/49R mutants in HEK293T cells. h Linear and K48 ubiquitination of β-Catenin immunoprecipitates in MDA-MB-231 cells treated with Dox (2 µg/ml), Gliotoxin (1 µM) or in combination for 24 h. i Linear and K48 ubiquitination of β-Catenin immunoprecipitates in HEK293T cells transfected with LUBAC and F-box-deleted β-TrCP mutant. j The analysis of β-catenin and β-TrCP interaction in MDA-MB-231 cells treated as in h. k Co-IP assay with anti-Flag antibody in HEK293T cells transfected with Flag-β-catenin WT, K133R, or K19/49 R. Source data are provided as a Source Data file.