Human HOIP/RNF31 Antibody

Detection of HOIP/RNF31 by Western Blot

Binding of OTULIN antagonizes SNX27 association with early endosomes. a Co-elution of endogenous OTULIN and SNX27 was analyzed by size exclusion chromatography. Upper panels: cell lysates of parental Jurkat T cells were fractionated using a Superdex 200 column and elution profiles of endogenous proteins (OTULIN, SNX27, HOIP, VPS35, and VPS26) were determined by WB. Lower panels: determination of elution profiles of endogenous SNX27 and OTULIN in OTULIN and SNX27 KO Jurkat T cells, respectively. Peak elution of molecular weight standards is depicted at the top. b HEK293 cells virally transduced with GFP-SNX27 (green) were stained for early endosomes using anti-EEA1 antibody (red) and co-localization was analyzed by confocal microscopy. c HEK293 cells were co-transduced with GFP-SNX27 (green) and RFP-OTULIN WT, delta ETSL or C129A (red). Localization of proteins was analyzed by confocal fluorescence microscopy. d HEK293 cells virally transduced with RFP-OTULIN WT, delta ETSL or C129A (gray) were stained for endogenous SNX27 (green) and EEA1 (red) and localization was analyzed by confocal fluorescence microscopy. e WT or OTULIN KO HEK293 cells were stained for endogenous SNX27 (green) and EEA1 (red) and localization was analyzed by confocal fluorescence microscopy. Co-localization in d and e was quantified by determining Pearson’s correlation using at least 12 pictures and imaging more than 100 cells for each condition. Graphs depict the mean ± SD. Two-tailed p-values: ns not significant, *p ≤ 0.05, ***p ≤ 0.001 by unpaired t-test. Scale bars: 10 µm. Source data are provided as a Source Data file Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/31541095), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of HOIP/RNF31 by Western Blot

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 delta ETSL (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, delta ETSL (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 delta ETSL. 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 Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/31541095), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human HOIP/RNF31 by Western Blot

A post-translational crosstalk controls stability of A20 and ABIN-1 after stimulation in Jurkat T cells. A Stability of ABIN-1 and A20 in Jurkat T cells after P/I stimulation treated with proteasome inhibitor MG132 (25 µM) analyzed by Western blot. B, C Stability of ABIN-1 and A20 in parental, CARD11 KO TRAF6 KO, HOIP KO and TRAF6/HOIP double KO Jurkat T cells after P/I stimulation analyzed by Western blot. D, E Stability of ABIN-1 and A20 in parental, ABIN-1 KO and A20 KO Jurkat T cells after CD3/CD28 and P/I stimulation analyzed by Western blot. F ABIN-1-IP and detection of A20-bound ABIN-1 in Jurkat T cells after P/I stimulation analyzed by Western blot. G ABIN-1 ubiquitination in HEK293 cells transfected with Flag-A20 WT, ZNF4/7 mt and OTU mt together with ABIN-1 and HA-K48Ub (K48-only) after ABIN-1-IP analyzed by Western blot. H A20 ubiquitination in HEK293 cells transfected with Flag-A20 WT, ZNF4/7 mt and OTU mt after HA-IP was analyzed by Western blot. I, J ABIN-1 stability and A20 cleavage in Jurkat T cells treated with MALT1 inhibitor MLT-985 (1 µM) following CD3/CD28 or P/I stimulation analyzed by Western blot Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35099607), licensed under a CC-BY license. Not internally tested by R&D Systems.