Fig. 7.

OTULIN counteracts SNX27 cargo loading. a Formation of a ternary HOIP-OTULIN-SNX27 complex was analyzed after co-expression of GFP-SNX27, HA-HOIP, and Flag-OTULIN in HEK293 cells. Ternary complex via Flag-OTULIN was verified using OTULIN Y56F (PIM mutation) and ΔETSL (PDZbm deletion) mutants. GFP-SNX27 was precipitated by GFP-Traps and association of OTULIN and HOIP was analyzed by WB. b SNX27 deficiency does not affect accumulation of Met1-ubiquitin chains. Extracts of WT, OTULIN-deficient, or SNX27-deficient HEK293 cells were analyzed for the abundance of Met1-ubiquitin chains and the expression of HOIP, OTULIN, and SNX27 by WB. c GFP or GFP-SNX27 constructs (WT or H114A) were expressed in HEK293 cells and cargo loading was analyzed by GFP-Traps by WB. d GFP-SNX27 WT was expressed in HEK293 cells alone or in the presence of HA-OTULIN WT, ΔETSL (PDZbm deletion), C129A (catalytically inactive), or C129A/L259E (catalytically inactive ubiquitin-binding mutant) and cargo loading was analyzed by GFP-Traps from cell lysates by WB. e Cargo-loading to endogenous SNX27 was assessed after anti-SNX27-IP in parental HEK293 cells as well as SNX27 KO or OTULIN KO cells. Binding of SNX27 to cargos and OTULIN was analyzed by WB. f Cargo-loading onto endogenous SNX27 was assessed by WB after anti-SNX27-IP in OTULIN KO HEK293 cells reconstituted with mock, OTULIN WT or OTULIN ΔETSL. Binding of each cargo to GFP-SNX27 (d) or SNX27 (e, f) was quantified from four independent experiments (see Supplementary Fig. 7a, b, e). A cumulative score for relative binding of all cargos to SNX27 was calculated and depicted as mean ± SD below the WB. Two-tailed p-values: ns not significant, ***p ≤ 0.001 by unpaired t-test. Source data are provided as a Source Data file