Fig 1: AMPKα and β subunits are substrates of LUBAC ubiquitination.A Immunoblots of FLAG-AMPKα1/2 IP from HEK293T cells co-transfected with LUBAC (HOIL-1 and HOIP), OTULIN-WT, catalytically inactive OTULIN-C129A (CA), or vector. B, C Immunoblots of M1-linked Ub conjugates purified from HEK293T cells transfected with LUBAC (HOIL-1, SHARPIN, and HOIP-WT or HOIP-C885S (CS)) (B) or LUBAC (HOIL-1 and HOIP) and OTULIN-WT or OTULIN-C129A (CA) (C) using recombinant GST-M1-Trap or GST-M1-SUB. D Immunoblots of in vitro ubiquitination assay (left) of HA-AMPKα1/2 IP from HEK293T cells (right) using recombinant E1, E2, LUBAC (HOIP, HOIL-1, and SHARPIN), and ubiquitin. All blots are representative of three (A, B, D) or two (C) independent experiments.
Fig 2: Proteomics identify LUBAC and OTULIN as interactors of AMPK.A Volcano plot of proteins from MS analysis of Strep-OTULIN affinity-purification (AP) from HEK293T cells versus vector control. Known OTULIN interactors (teal) and AMPK pathway proteins (orange) highlighted. False discovery rate (FDR) < 0.05; Fold-change (FC) cutoff 2.5. B Immunoblots of Strep-OTULIN AP from HEK293T cells transfected with 2xHA-2xStrep-OTULIN or vector. C Immunoblots of Strep-HOIP AP from HEK293T cells co-transfected with V5-HOIL-1, V5-SHARPIN, and either 2xHA-2xStrep-HOIP or vector. Immunoblots of endogenous HOIP (D) or OTULIN (E) immunoprecipitation (IP) from WT, HOIP-KO, and OTULIN-KO U2OS cells. F Top: Schematic representation of HOIP variants, including full-length wild-type (FL-WT), truncated mutants (PUB-UBA, PUB-NZFs, PUB, RBR-LDD), and the catalytically inactive mutant (FL-C885S). Bottom: Immunoblots of HA-tagged HOIP variants AP from HEK293T cells transfected with V5-HOIL-1, V5-SHARPIN, and 2xHA-2xStrep-HOIP or vector. G Left: Immunoblots of Strep-OTULIN AP from HEK293T cells transfected with 2xHA-2xStrep-OTULIN variants or vector. Right: Schematic of OTULIN WT, ΔPBM, Y56A, and ΔN mutants. H Volcano plot of proteins from MS analysis of Strep-AMPKα1 (PRKAA1) AP from HEK293T cells versus vector control. AMPK subunits (PRKAB1/2 and PRKAG1) (orange) and OTULIN and HOIP (RNF31) (blue) are highlighted. FDR < 0.05; FC cutoff 3.7. I Immunoblots of endogenous AMPKα1/2 IP from U2OS WT or AMPKα1/2 double-KO (DKO) cells. J Immunoblots of FLAG-AMPKα1 or α2 IP from HEK293T cells transfected with vector, 2xFLAG-2xStrep-AMPKα1, or α2. All blots are representative of three independent experiments.
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