rabbit polyclonal anti ube2d3 antibody  (Proteintech)

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A ) Schematic showing the design of dual-gRNA directed CRISPR screen of E2s regulating the CM-induced degradation of ePL-tagged IKZF1 in 384-well array format. ( B ) Chemiluminescent measurement of ePL-IKZF1 protein expression level in U937_Cas9_ePL-IKZF1 parental cells or cells expressing UBE2G1 -specfic sgRNA alone or in combination with non-targeting or UBE2D3 -specific sgRNA. Cells were treated with POM at the indicated concentrations for 16 hr. Data are presented as mean ± SD, n = 4 technical replicates. ( C ) Immunoblot analysis of U937-Cas9 parental cells or cells expressing non-targeting sgRNA, UBE2G1 -specific sgRNA, UBE2D3 -specfic sgRNA, or both UBE2G1 and UBE2D3 sgRNAs. Cells were treated with POM at the indicated concentrations for 16 hr. SE, short exposure; LE, long exposure. Result is representative of three independent experiments. 10.7554/eLife.40958.012 Figure 2—source data 1. ePL luminescence signal shown in .

Article Snippet: Antibody , anti-UBE2D3 (rabbit polyclonal antibody) , Sigma-Aldrich , RRID: AB_10605875 , WB (1: 1,000).

Techniques: CRISPR, Expressing, Western Blot

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A ) Sequence alignment of human UBE2G1, human UBE2G2 and human CDC34 using Clustal W 2.1. The acidic loops indispensable for the assembly of K48-linked ubiquitin chains are highlighted with red, and the catalytic cysteines are highlighted with blue. ( B ) Immunoblot analysis of 293T parental or UBE2G1-/- cells transduced with lentiviral vectors expressing FLAG-tagged UBE2G1 wild-type or C90S mutant, or FLAG-tagged UBE2D3 wild-type or C85S mutant. Cells were treated with CC-885 at the indicated concentrations for 4 hr. Note that overexpression of wild-type FLAG-UBE2G1 or FLAG-UBE2D3 partially rescued the GSPT1 degradation defect caused by UBE2G1 deficiency, while overexpression of catalytically-dead mutant FLAG-UBE2G1-C90S or FLAG-UBE2D3-C85S further blocked the degradation of GSPT1. ( C ) In vitro ubiquitination of GSPT1 by CRL4 CRBN with or without CC-885 and indicated E2 variants. Consistent with results observed with bacterial recombinant UBE2G1 and UBE2D3 proteins, FLAG-UBE2G1 and FLAG-UBE2D3 proteins purified from human cells acted in concert to promote the ubiquitination of GSPT1. Results shown in ( B ) and ( C ) are representative of three independent experiments.

Article Snippet: Antibody , anti-UBE2D3 (rabbit polyclonal antibody) , Sigma-Aldrich , RRID: AB_10605875 , WB (1: 1,000).

Techniques: Sequencing, Western Blot, Transduction, Expressing, Mutagenesis, Over Expression, In Vitro, Recombinant, Purification

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A– D ) In vitro ubiquitination of IKZF1 ( A and C ) and GSPT1 ( B and D ) MBP fusion proteins by recombinant CRL4 CRBN complex. Recombinant protein products as indicated were incubated with or without 80 µM POM ( A and C ) or 80 µM CC-885 ( B and D ) in the ubiquitination assay buffer containing 80 mM ATP at 30°C for 2 hr, and then analyzed by immunoblotting. ( E ) Sequential in vitro ubiquitination of GSPT1 by recombinant CRL4 CRBN complex. MBP-GSPT1 recombination protein was incubated with Ube1, UBE2D3, Cul4-Rbx1, DDB1-cereblon, Ubiquitin, ATP and CC-885 in the ubiquitination assay at 30°C for 4 hr. After purification over size-exclusion chromatography, pre-ubiquitinated MBP-GSPT1 protein was then incubated with Ube1, DDB1-cereblon, Ubiquitin, ATP and UBE2G1 with or without CC-885 or Cul4A-Rbx1 in the ubiquitination assay at 30°C for 2 hr, followed by immunoblot analysis. ( F ) Schematic showing the sequential ubiquitination of CRBN neomorphic substrates by UBE2D3 and UBE2G1. Results shown in ( A–E ) are representative of three independent experiments.

Article Snippet: Antibody , anti-UBE2D3 (rabbit polyclonal antibody) , Sigma-Aldrich , RRID: AB_10605875 , WB (1: 1,000).

Techniques: In Vitro, Recombinant, Incubation, Ubiquitin Assay, Western Blot, Purification, Size-exclusion Chromatography

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A and B ) 293T parental and UBE2G1-/-;UBE2D3-/- ( clone 4) cells were transiently transfected with plasmids expressing cereblon, V5-tagged IKZF1 and 8xHis-Ub with or without UBE2G1, UBE2D3 or both. ( C ) 293T parental and UBE2G1-/- (clone 13) cells were transiently transfected with plasmids expressing cereblon, IKZF1-V5, 8xHis-Ub with or without UBE2G1 wild-type or C90S mutant. In ( A ), ( B ) and ( C ), 48 hr after transfection, cells were treated with MG-132 (10 µM) and POM at the indicated concentrations for additional 8 hr. Ubiquitinated protein products enriched with magnetic nickel sepharose were subjected to immunoblot analysis. Immunoblot analysis of whole cell extracts showing equal input proteins is shown in . All results shown in this figure are representative of three independent experiments.

Article Snippet: Antibody , anti-UBE2D3 (rabbit polyclonal antibody) , Sigma-Aldrich , RRID: AB_10605875 , WB (1: 1,000).

Techniques: Transfection, Expressing, Mutagenesis, Western Blot

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A ) Total input for . Immunoblot analysis of 293T parental and UBE2G1-/-;UBE2D3-/- (Clone 4) cells transfected to produce 8xHis-Ubiquitin, cereblon and IKZF1-V5. ( B ) Total input for . Immunoblot analysis of 293T parental and UBE2G1-/-;UBE2D3-/- (Clone 4) cells transfected to produce 8xHis-Ubiquitin, CRBN, IKZF1-V5 with or without UBE2G1 and/or UBE2D3. ( C ) Total input for . Immunoblot analysis of 293T parental and UBE2G1-/- (Clone 13) cells transfected to produce 8xHis-Ubiquitin, CRBN, IKZF1-V5 with or without UBE2G1 wildtype or C90S mutant. In ( A ), ( B ) or ( C ), 48 hr after transfection, cells were treated with 10 µM MG-132 and POM at the indicated concentrations for additional 8 hr. All results shown in this figure are representative of three independent experiments.

Article Snippet: Antibody , anti-UBE2D3 (rabbit polyclonal antibody) , Sigma-Aldrich , RRID: AB_10605875 , WB (1: 1,000).

Techniques: Western Blot, Transfection, Mutagenesis

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A ) Sequence alignment of human UBE2D family proteins using Clustal W 2.1. Note that the amino acid sequence identity among all four family proteins is close to 90%. ( B ) In vitro ubiquitination of GSPT1 MBP fusion protein by recombinant CRL4 CRBN complex in the presence of UBE2G1, UBE2D1, UBE2D2, or UBE2D3, alone or in combination. Recombinant protein products as indicated were incubated with or without 80 µM CC-885 in the ubiquitination assay buffer at 30°C for 2 hr, and then analyzed by immunoblotting. SE, short exposure; LE, long exposure. Result is representative of three independent experiments.

Article Snippet: Antibody , anti-UBE2D3 (rabbit polyclonal antibody) , Sigma-Aldrich , RRID: AB_10605875 , WB (1: 1,000).

Techniques: Sequencing, In Vitro, Recombinant, Incubation, Ubiquitin Assay, Western Blot

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet:

Article Snippet: Antibody , anti-UBE2D3 (rabbit polyclonal antibody) , Sigma-Aldrich , RRID: AB_10605875 , WB (1: 1,000).

Techniques: Recombinant, Plasmid Preparation, Transfection, Transduction, Magnetic Beads