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( A ) qPCR analysis of Cebpb mRNA expression in control and Peli1 -knockdown BV2 cells. ( B ) Immunoblot of C/EBPβ and Hsp60 (loading control) in control and Peli1 -knockdown BV2 cells that pretreated with or without a proteasome inhibitor MG132 for 4 hours. ( C ) Immunofluorescent images showing the intracellular colocalization of Peli1 and C/EBPβ in primary microglia and BV2 cells. The zoomed images indicated the colocalization of Peli1 and C/EBPβ in the nucleus of the cells. Scale bars: 10 μm. ( D ) Immunoassays on lysates of BV2 cells after immunoprecipitation with control IgG or anti-C/EBPβ and immunoblot analysis of C/EBPβ associated Peli1. ( E, F ) Ubiquitination of endogenous C/EBPβ in Peli1 +/− and Peli1 −/− primary microglia ( E ) and in control and Peli1 -knockdown BV2 cells ( F ) that were pretreated with MG132 for 4 hours, assessed by immunoblot analysis with anti-ubiquitin and anti-C/EBPβ after immunoprecipitation with anti-C/EBPβ (top), and by immunoblot analysis with input proteins and loading control (below). ( G ) Ubiquitination of C/EBPβ in 293T cells transfected with the indicated expression vectors, assessed by immunoblot analysis with anti-HA after immunoprecipitation with anti-C/EBPβ (top) or by immunoblot analysis with input proteins in lysates without immunoprecipitation (below). ( H ) In vitro ubiquitination assay of C/EBPβ ubiquitination after a mixture reaction of ubiquitin-charged E2 <t>UbcH5a,</t> in vitro translated C/EBPβ, and with or without HA-Peli1 or HA-Peli1ΔC proteins. Data with error bars represent mean ± SEM. Each panel is representative of at least 3 independent experiments. Numerical values for (A) are available in . C/EBP, CCAAT/enhancer-binding protein; HA, hemagglutinin; IB, immunoblot; IgG, immunoglobulin G; IP, immunoprecipitation; Peli1ΔC, C-terminal-deleted Peli1; qPCR, quantitative PCR; UbcH5a, ubiquitin-conjugating enzyme H5a.
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ubiquitin-conjugating enzyme ubch5a  (Boston Biochem)
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( A ) qPCR analysis of Cebpb mRNA expression in control and Peli1 -knockdown BV2 cells. ( B ) Immunoblot of C/EBPβ and Hsp60 (loading control) in control and Peli1 -knockdown BV2 cells that pretreated with or without a proteasome inhibitor MG132 for 4 hours. ( C ) Immunofluorescent images showing the intracellular colocalization of Peli1 and C/EBPβ in primary microglia and BV2 cells. The zoomed images indicated the colocalization of Peli1 and C/EBPβ in the nucleus of the cells. Scale bars: 10 μm. ( D ) Immunoassays on lysates of BV2 cells after immunoprecipitation with control IgG or anti-C/EBPβ and immunoblot analysis of C/EBPβ associated Peli1. ( E, F ) Ubiquitination of endogenous C/EBPβ in Peli1 +/− and Peli1 −/− primary microglia ( E ) and in control and Peli1 -knockdown BV2 cells ( F ) that were pretreated with MG132 for 4 hours, assessed by immunoblot analysis with anti-ubiquitin and anti-C/EBPβ after immunoprecipitation with anti-C/EBPβ (top), and by immunoblot analysis with input proteins and loading control (below). ( G ) Ubiquitination of C/EBPβ in 293T cells transfected with the indicated expression vectors, assessed by immunoblot analysis with anti-HA after immunoprecipitation with anti-C/EBPβ (top) or by immunoblot analysis with input proteins in lysates without immunoprecipitation (below). ( H ) In vitro ubiquitination assay of C/EBPβ ubiquitination after a mixture reaction of ubiquitin-charged E2 <t>UbcH5a,</t> in vitro translated C/EBPβ, and with or without HA-Peli1 or HA-Peli1ΔC proteins. Data with error bars represent mean ± SEM. Each panel is representative of at least 3 independent experiments. Numerical values for (A) are available in . C/EBP, CCAAT/enhancer-binding protein; HA, hemagglutinin; IB, immunoblot; IgG, immunoglobulin G; IP, immunoprecipitation; Peli1ΔC, C-terminal-deleted Peli1; qPCR, quantitative PCR; UbcH5a, ubiquitin-conjugating enzyme H5a.
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ubiquitin-conjugating enzyme ubch5a - by Bioz Stars, 2026-09
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( A ) qPCR analysis of Cebpb mRNA expression in control and Peli1 -knockdown BV2 cells. ( B ) Immunoblot of C/EBPβ and Hsp60 (loading control) in control and Peli1 -knockdown BV2 cells that pretreated with or without a proteasome inhibitor MG132 for 4 hours. ( C ) Immunofluorescent images showing the intracellular colocalization of Peli1 and C/EBPβ in primary microglia and BV2 cells. The zoomed images indicated the colocalization of Peli1 and C/EBPβ in the nucleus of the cells. Scale bars: 10 μm. ( D ) Immunoassays on lysates of BV2 cells after immunoprecipitation with control IgG or anti-C/EBPβ and immunoblot analysis of C/EBPβ associated Peli1. ( E, F ) Ubiquitination of endogenous C/EBPβ in Peli1 +/− and Peli1 −/− primary microglia ( E ) and in control and Peli1 -knockdown BV2 cells ( F ) that were pretreated with MG132 for 4 hours, assessed by immunoblot analysis with anti-ubiquitin and anti-C/EBPβ after immunoprecipitation with anti-C/EBPβ (top), and by immunoblot analysis with input proteins and loading control (below). ( G ) Ubiquitination of C/EBPβ in 293T cells transfected with the indicated expression vectors, assessed by immunoblot analysis with anti-HA after immunoprecipitation with anti-C/EBPβ (top) or by immunoblot analysis with input proteins in lysates without immunoprecipitation (below). ( H ) In vitro ubiquitination assay of C/EBPβ ubiquitination after a mixture reaction of ubiquitin-charged E2 <t>UbcH5a,</t> in vitro translated C/EBPβ, and with or without HA-Peli1 or HA-Peli1ΔC proteins. Data with error bars represent mean ± SEM. Each panel is representative of at least 3 independent experiments. Numerical values for (A) are available in . C/EBP, CCAAT/enhancer-binding protein; HA, hemagglutinin; IB, immunoblot; IgG, immunoglobulin G; IP, immunoprecipitation; Peli1ΔC, C-terminal-deleted Peli1; qPCR, quantitative PCR; UbcH5a, ubiquitin-conjugating enzyme H5a.
E2 Ubiquitin Conjugating Enzymes (Ubch5a), supplied by Boston Biochem, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews
e2 ubiquitin-conjugating enzymes (ubch5a) - by Bioz Stars, 2026-09
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( A ) qPCR analysis of Cebpb mRNA expression in control and Peli1 -knockdown BV2 cells. ( B ) Immunoblot of C/EBPβ and Hsp60 (loading control) in control and Peli1 -knockdown BV2 cells that pretreated with or without a proteasome inhibitor MG132 for 4 hours. ( C ) Immunofluorescent images showing the intracellular colocalization of Peli1 and C/EBPβ in primary microglia and BV2 cells. The zoomed images indicated the colocalization of Peli1 and C/EBPβ in the nucleus of the cells. Scale bars: 10 μm. ( D ) Immunoassays on lysates of BV2 cells after immunoprecipitation with control IgG or anti-C/EBPβ and immunoblot analysis of C/EBPβ associated Peli1. ( E, F ) Ubiquitination of endogenous C/EBPβ in Peli1 +/− and Peli1 −/− primary microglia ( E ) and in control and Peli1 -knockdown BV2 cells ( F ) that were pretreated with MG132 for 4 hours, assessed by immunoblot analysis with anti-ubiquitin and anti-C/EBPβ after immunoprecipitation with anti-C/EBPβ (top), and by immunoblot analysis with input proteins and loading control (below). ( G ) Ubiquitination of C/EBPβ in 293T cells transfected with the indicated expression vectors, assessed by immunoblot analysis with anti-HA after immunoprecipitation with anti-C/EBPβ (top) or by immunoblot analysis with input proteins in lysates without immunoprecipitation (below). ( H ) In vitro ubiquitination assay of C/EBPβ ubiquitination after a mixture reaction of ubiquitin-charged E2 UbcH5a, in vitro translated C/EBPβ, and with or without HA-Peli1 or HA-Peli1ΔC proteins. Data with error bars represent mean ± SEM. Each panel is representative of at least 3 independent experiments. Numerical values for (A) are available in . C/EBP, CCAAT/enhancer-binding protein; HA, hemagglutinin; IB, immunoblot; IgG, immunoglobulin G; IP, immunoprecipitation; Peli1ΔC, C-terminal-deleted Peli1; qPCR, quantitative PCR; UbcH5a, ubiquitin-conjugating enzyme H5a.

Journal: PLoS Biology

Article Title: Peli1 impairs microglial Aβ phagocytosis through promoting C/EBPβ degradation

doi: 10.1371/journal.pbio.3000837

Figure Lengend Snippet: ( A ) qPCR analysis of Cebpb mRNA expression in control and Peli1 -knockdown BV2 cells. ( B ) Immunoblot of C/EBPβ and Hsp60 (loading control) in control and Peli1 -knockdown BV2 cells that pretreated with or without a proteasome inhibitor MG132 for 4 hours. ( C ) Immunofluorescent images showing the intracellular colocalization of Peli1 and C/EBPβ in primary microglia and BV2 cells. The zoomed images indicated the colocalization of Peli1 and C/EBPβ in the nucleus of the cells. Scale bars: 10 μm. ( D ) Immunoassays on lysates of BV2 cells after immunoprecipitation with control IgG or anti-C/EBPβ and immunoblot analysis of C/EBPβ associated Peli1. ( E, F ) Ubiquitination of endogenous C/EBPβ in Peli1 +/− and Peli1 −/− primary microglia ( E ) and in control and Peli1 -knockdown BV2 cells ( F ) that were pretreated with MG132 for 4 hours, assessed by immunoblot analysis with anti-ubiquitin and anti-C/EBPβ after immunoprecipitation with anti-C/EBPβ (top), and by immunoblot analysis with input proteins and loading control (below). ( G ) Ubiquitination of C/EBPβ in 293T cells transfected with the indicated expression vectors, assessed by immunoblot analysis with anti-HA after immunoprecipitation with anti-C/EBPβ (top) or by immunoblot analysis with input proteins in lysates without immunoprecipitation (below). ( H ) In vitro ubiquitination assay of C/EBPβ ubiquitination after a mixture reaction of ubiquitin-charged E2 UbcH5a, in vitro translated C/EBPβ, and with or without HA-Peli1 or HA-Peli1ΔC proteins. Data with error bars represent mean ± SEM. Each panel is representative of at least 3 independent experiments. Numerical values for (A) are available in . C/EBP, CCAAT/enhancer-binding protein; HA, hemagglutinin; IB, immunoblot; IgG, immunoglobulin G; IP, immunoprecipitation; Peli1ΔC, C-terminal-deleted Peli1; qPCR, quantitative PCR; UbcH5a, ubiquitin-conjugating enzyme H5a.

Article Snippet: The ubiquitination reaction was processed by mixing the translated proteins with precharged ubiquitin-conjugating enzyme E2 UbcH5a (E2-800, Boston Biochem) and incubating at 37°C for 4 hours according to the manufacturer’s protocol.

Techniques: Expressing, Control, Knockdown, Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Transfection, In Vitro, Binding Assay, Real-time Polymerase Chain Reaction