ube1 e-305  (Boston Biochem)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Ebastine-mediated destabilization of E3 ligase MKRN1 protects against metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00018-024-05535-2

Figure Lengend Snippet: Ebastine induces MKRN1 destabilization. A HepG2 cells were treated with 0, 5, or 10 μM ebastine for 16 h or 24 h followed by western blotting. B HepG2 cells were treated with 10 μM ebastine for 24 h in the presence or absence of 20 μM MG132 for 6h followed by western blotting. C The MKRN1 mRNA levels were analyzed via qRT-PCR in the presence or absence of ebastine in HepG2 cells (n = 3 per group). D 293T cells were transfected with plasmids expressing HA/MKRN1 WT, HA/MKRN1 H307E mutant, and HA/Ub with or without ebastine. After 18 h, the transfected cells were treated with MG132 for 6h in the presence or absence of ebastine. Cell lysates were immunoprecipitated using anti-MKRN1 antibodies followed by western blotting using anti-Ub antibodies. E Ubiquitinated endogenous MKRN1 was determined under denaturing conditions using MG132-treated HepG2 cells. Immunoprecipitation of cell lysates with anti-MKRN1 antibodies was followed by western blotting with anti-Ub antibodies. F Schematic illustrations of MKRN1 WT and its mutants. G 293T cells were transfected with plasmids expressing HA/MKRN1 WT and its mutants followed by ebastine treatment. H In vitro ubiquitination analysis of recombinant MKRN1 C-terminus (264–482) in the presence or absence of E1, E2, Ub, ATP, or ebastine. I 293T cells were transfected with plasmids expressing HA/MKRN1 WT or HA/MKRN1 AA mutant followed by ebastine treatment. J 293T cells were transfected with plasmids expressing HA/MKRN1 WT, HA/MKRN1 AA mutant, and HA/Ub with or without ebastine. After 18 h, the transfected cells were treated with MG132 for 6h in the presence or absence of ebastine. Cell lysates were immunoprecipitated using anti-MKRN1 antibodies followed by western blotting using anti-HA antibodies. One-way ANOVA with Dunnett’s multiple comparison test; n.s. not significant

Article Snippet: For in vitro ubiquitination assays, a 0.5 μg sample of recombinant proteins purified using bacterial systems was incubated with 100 ng of E1 (UBE1, E-305, Boston Biochem, Cambridge, MA, USA), 250 ng of E2 (UbcH5c, E2-627, Boston Biochem), and 5 μg of ubiquitin (U-100H, Boston Biochem) in 20 μl of reaction buffer (40 mM of Tris, 50 mM of NaCl, 5 mM of MgCl 2 , 2 mM of ATP and 1 mM of dithiothreitol, pH 7.6) as indicated.

Techniques: Western Blot, Quantitative RT-PCR, Transfection, Expressing, Mutagenesis, Immunoprecipitation, In Vitro, Ubiquitin Proteomics, Recombinant, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Ebastine-mediated destabilization of E3 ligase MKRN1 protects against metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00018-024-05535-2

Figure Lengend Snippet: Ebastine prevents MKRN1-mediated ubiquitination and degradation of AMPKα2. A , B HepG2 cells treated with DMSO, ebastine, or siMKRN1 were incubated in the absence (top) or presence (bottom) of compound C with BSA or 1 mM OPA. Representative fluorescence microscopy images of HepG2 cells stained with Hoechst and Nile red (Scale bars = 10 µm) ( A ). Analysis of the relative fluorescence intensity of OPA-treated cells ( B ). C HepG2 cells were treated with 0, 5, or 10 μM ebastine for 16 h or 24 h followed by western blotting. D 293T cells were transfected with plasmids expressing FLAG/AMPKα2 and/or HA/MKRN1 with or without ebastine followed by western blotting. E 293T cells were transfected with plasmids expressing MKRN1 WT, FLAG/AMPKα2, and HA/Ub in the presence or absence of ebastine. Cell lysates were immunoprecipitated using anti-FLAG antibodies followed by western blotting using anti-HA antibodies. F HA/Ub-expressing plasmid was transfected into HepG2 cells in the presence or absence of ebastine. Immunoprecipitation of cell lysates with anti-AMPKα2 antibodies was followed by western blotting with anti-HA antibodies. G , H Representative fluorescence microscopy images of WT or MK1 −/− MEFs stained with Hoechst and Nile red after treatment with BSA or 1 mM OPA and DMSO or ebastine ( G ), and quantitative analysis of the relative fluorescence intensity of OPA-treated cells (Scale bars = 10 µm) ( H ). I WT or MK1 −/− MEFs were treated with ebastine followed by western blotting. At least three independent experiments were performed, with a minimum of 100 cells analyzed per group for quantitative analysis. One-way ANOVA with Dunnett’s multiple comparison test; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, n.s. not significant

Article Snippet: For in vitro ubiquitination assays, a 0.5 μg sample of recombinant proteins purified using bacterial systems was incubated with 100 ng of E1 (UBE1, E-305, Boston Biochem, Cambridge, MA, USA), 250 ng of E2 (UbcH5c, E2-627, Boston Biochem), and 5 μg of ubiquitin (U-100H, Boston Biochem) in 20 μl of reaction buffer (40 mM of Tris, 50 mM of NaCl, 5 mM of MgCl 2 , 2 mM of ATP and 1 mM of dithiothreitol, pH 7.6) as indicated.

Techniques: Ubiquitin Proteomics, Incubation, Fluorescence, Microscopy, Staining, Western Blot, Transfection, Expressing, Immunoprecipitation, Plasmid Preparation, Comparison