Journal: eLife

Article Title: Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger cyclin K degradation

doi: 10.7554/eLife.59994

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-UBE2G1 (Rabbit polyclonal) , Proteintech(12012–1-AP) , RRID: AB_10665812 , WB (1:2000).

Techniques: Recombinant, Amplified Luminescent Proximity Homogenous Assay, Software

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 the CRISPR screen to identify E2 enzyme(s) 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 non-targeting or UBE2G1 -specific sgRNAs. 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 or UBE2G1-/- cells with or without stable expression of UBE2G1 wild-type or C90S mutant. Cells were treated with POM at the indicated concentrations for 16 hr. Result is representative of three independent experiments. 10.7554/eLife.40958.008 Figure 1—source data 1. ePL luminescence signal shown in .

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: CRISPR, Expressing, Western Blot, Mutagenesis

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: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

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–U ) Chemiluminescent measurement of ePL-IKZF1 protein expression level in U937_Cas9_ePL-IKZF1 parental cells or cells expressing both UBE2G1 -specific and non-targeting or E2-specific sgRNA. Cells were treated with DMSO or an increasing concentrations of POM for 16 hr. Data are presented as mean ± SD, n = 4 technical replicates. 10.7554/eLife.40958.011 Figure 2—figure supplement 1—source data 1. ePL luminescence signal shown in .

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: Expressing

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A and B ) Immunoblot analysis of OPM2-Cas9 cells expressing non-targeting, UBE2G1 -specific or CRBN -specific sgRNA. Cells were treated with LEN, POM or CC-220 for 16 hr ( A ) or CC-885 for 4 hr ( B ) at the indicated concentrations. ( C ) Immunoblot analysis of 293T parental or UBE2G1-/- cells treated with BRD4 PROTACs dBET1 or MZ1 at the indicated concentrations for 16 hr. ( D ) Immunoblot analysis of OPM2 parental or UBE2G1-/- cells treated with 100 µg/ml cycloheximide with or without 1 µM POM pretreatment for half an hour. Cells were harvested at the indicated time points. All results shown in this figure are representative of three independent experiments.

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: Western Blot, Expressing

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A–F ) Immunoblot analysis of DF15-Cas9 cells ( A and C ), MM1S-Cas9 cells ( B and D ), OCI-AML2-Cas9 cells ( E ), U937-Cas9 cells ( F ), MOLM-13-Cas9 cells ( G ) and MV4-11-Cas9 cells ( H ) transduced with lentiviral vectors expressing non-targeting, UBE2G1 -specific or CRBN -specific sgRNAs. Cells were treated with lenalidomide for 16 hr ( A and B ), or CC-885 for 4 hr ( C–H ) at the indicated concentrations. ( I ) Immunoblot analysis of 293T parental or UBE2G1-/- cells stably expressing UBE2G1 wild-type or C90S mutant. Cells were treated with CC-885 at the indicated concentrations for 4 hr. All results shown in this figure are representative of three independent experiments.

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: Western Blot, Transduction, Expressing, Stable 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 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: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: Sequencing, Ubiquitin Proteomics, 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: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: In Vitro, Ubiquitin Proteomics, Recombinant, Incubation, 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: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

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: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: Western Blot, Transfection, Ubiquitin Proteomics, Mutagenesis

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A ) Effect of LEN (top panel) and POM (bottom panel) on proliferation of myeloma cell lines. Cell proliferation was determined by CTG. Data are presented as mean ± SD, n = 3 technical replicates. ( B ) Immunoblot analysis of myeloma cell lines used in ( A ). ( C and D ) Proliferation ( C ) and immunoblot analysis ( D ) of SKMM2 cells transduced with lentiviral vectors encoding GFP, UBE2G1 and UBE2G1-C90S. Cells were treated with DMSO vehicle control, LEN or POM at the indicated concentrations for 5 days ( C ) or 16 hr ( D ). In ( C ), cell proliferation was determined by CTG, and data are presented as mean ± SD, n = 3 technical replicates. All results shown in this figure are representative of three independent experiments. 10.7554/eLife.40958.024 Figure 6—source data 1. CTG luminescence signal shown in .

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: Western Blot, Transduction, Control

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A–D ) Cell proliferation of AML cell lines OCI-AML2 ( A ), U937 ( B ), MOLM-13 ( C ) and MV4-11 ( D ) treated with DMSO or CC-885 at the indicated concentrations for 72 hr. Cell proliferation was determined by CTG. Data are presented as mean ± SD, n = 4 technical replicates. ( E and F ) Cell proliferation of 293T parental and UBE2G1-/- (clone 13) cells with or without ectopic overexpression of UBE2G1 wild-type or C90S mutant. Cells were treated with CC-885 ( E ), dBET1 ( F ) or MZ-1 ( F ). Cell proliferation was determined by CTG. Data are presented as mean ± SD, n = 4 technical replicates. All results shown in this figure are representative of three independent experiments. 10.7554/eLife.40958.023 Figure 6—figure supplement 1—source data 1. CTG luminescence signal shown in .

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: Over Expression, Mutagenesis

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A and B ) Cell proliferation ( A ) and immunoblot analysis ( B ) of OPM2-Cas9 cells transduced with lentiviral vectors expressing non-targeting, UBE2G1 -specific or CRBN -specific sgRNAs. Cells were treated DMSO vehicle control, LEN, POM or CC-220 at the indicated concentrations for 5 days ( A ) or 16 hr ( B ). In ( A ), cell proliferation was determined by CTG, and data are presented as mean ± SD, n = 3 technical replicates. All results shown in this figure are representative of three independent experiments. 10.7554/eLife.40958.028 Figure 7—source data 1. CTG luminescence signal shown in .

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: Western Blot, Transduction, Expressing, Control

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A–D ) Cell proliferation ( A and C ) and immunoblot analysis ( B and D ) of MM1S-Cas9 ( A and B ) and DF15-Cas9 ( C and D ) cells infected with lentiviral vectors expressing non-targeting, UBE2G1 -specific or CRBN -specific sgRNAs. Cells were treated DMSO vehicle control, LEN, POM or CC-220 at the indicated concentrations for 5 days ( A and C ) or 16 hr ( B and D ). In ( A ) and ( C ), cell proliferation was determined by CTG, and data are presented as mean ± SD, n = 3 technical replicates. All results shown in this figure are representative of three independent experiments. 10.7554/eLife.40958.027 Figure 7—figure supplement 1—source data 1. CTG luminescence signal shown in .

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: Western Blot, Infection, Expressing, Control

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A and B ) Immunoblot analysis of 293T parental and UBE2G1-/- (clone 13) cells treated with UV irradiation ( A ) or E7070 ( B ). In ( A ), cells were UV irradiated at 50 J/m2 using a Stratalinker, and collected at the indicated time points thereafter. In ( B ), cells were treated with DMSO or an increasing concentration of E7070 for 16 hr. All results shown in this figure are representative of three independent experiments.

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: Western Blot, Irradiation, Concentration Assay

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: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

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

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet:

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

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

Journal: International Journal of Molecular Sciences

Article Title: UBE2G1 Is a Critical Component of Immune Response to the Infection of Pseudomonas Plecoglossicida in Large Yellow Croaker ( Larimichthys crocea )

doi: 10.3390/ijms23158298

Figure Lengend Snippet: LcUbe2g1 gene sequence and the tertiary structure of Lc UBE2G1 protein. ( A ) The coding sequence of LcUbe2g1 and deduced amino acid sequence. The stop codons were indicated with asterisk (*), the UBCc (ubiquitin-conjugating catalytic) domain fold was underlined, the blue shade indicate the ubiquitin (Ub) binding motifs (HPN), the red shade indicate the active site cysteine (C), the purple boxes indicate the Ser phosphorylation sites, the green boxes indicate Thr phosphorylation sites and the blue boxes indicate Tyr phosphorylation sites; ( B ) Tertiary structure of Lc UBE2G1 protein. Lc UBE2G1 contains a UBC core catalytic domain consisting of 4α helices and 4β sheets, the purple spheres represent a conserved HPN pattern (His81, Pro82, Asn83) and the red spheres indicate a cysteine (Cys91) active site.

Article Snippet: The antibody incubation steps are as follows: firstly, the blocked cells were incubated with ubiquitin protein antibody (Mouse; 1:100; Abcam; Cambridge, MA, USA) and UBE2G1 protein antibody (Rabbit; 1:300; Irvine, Gentex Inc., Zeeland, MI, USA) at 37°C for 1 h; then incubated with Alexa Fluor 488-labeled (green) goat anti-rabbit, Alexa Fluor 555-labeled (red) donkey anti-mouse fluorescent secondary antibody (1:500; Beyotime, Shanghai, China) for an additional 1 h at 37 °C.

Techniques: Sequencing, Ubiquitin Proteomics, Binding Assay, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: UBE2G1 Is a Critical Component of Immune Response to the Infection of Pseudomonas Plecoglossicida in Large Yellow Croaker ( Larimichthys crocea )

doi: 10.3390/ijms23158298

Figure Lengend Snippet: LcUbe2g1 tissue expression profile and the subcellular localization of Lc UBE2G1. ( A ) Relative transcript levels of LcUbe2g1 in different tissues. RT-qPCR was performed to determine the relative expression of LcUbe2g1 in different tissues of Larimichthys crocea , including brain, kidney, gills, head kidney, spleen, liver, fins and intestine. β -actin was used as an internal control to normalize the expression level; ( B ) Subcellular localization of Lc UBE2G1 protein in transfected Larimichthys crocea head kidney cells. The images were captured under confocal fluorescence microscopy. Scale bar = 50 µm.

Article Snippet: The antibody incubation steps are as follows: firstly, the blocked cells were incubated with ubiquitin protein antibody (Mouse; 1:100; Abcam; Cambridge, MA, USA) and UBE2G1 protein antibody (Rabbit; 1:300; Irvine, Gentex Inc., Zeeland, MI, USA) at 37°C for 1 h; then incubated with Alexa Fluor 488-labeled (green) goat anti-rabbit, Alexa Fluor 555-labeled (red) donkey anti-mouse fluorescent secondary antibody (1:500; Beyotime, Shanghai, China) for an additional 1 h at 37 °C.

Techniques: Expressing, Quantitative RT-PCR, Control, Transfection, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: UBE2G1 Is a Critical Component of Immune Response to the Infection of Pseudomonas Plecoglossicida in Large Yellow Croaker ( Larimichthys crocea )

doi: 10.3390/ijms23158298

Figure Lengend Snippet: Defense response of UBE2G1 against P.plecoglossicid . ( A ) Time course expression of LcUbe2g1 in different tissues after P.plecoglossicid infection. The relative expression levels of LcUbe2g1 were determined by RT-qPCR in kidney, liver and spleen at 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h and 120 h after P. plecoglossicid challenge. β-actin was used as an internal control. Data are mean ± SE ( n = 6 or 3). The letters a, b, c, d represent statistical significance ( p < 0.05); ( B ) Immunohistochemical staining of UBE2G1 protein in spleen, kidney, liver and brain of large yellow croaker. Positive signal was indicated with brown stain.

Article Snippet: The antibody incubation steps are as follows: firstly, the blocked cells were incubated with ubiquitin protein antibody (Mouse; 1:100; Abcam; Cambridge, MA, USA) and UBE2G1 protein antibody (Rabbit; 1:300; Irvine, Gentex Inc., Zeeland, MI, USA) at 37°C for 1 h; then incubated with Alexa Fluor 488-labeled (green) goat anti-rabbit, Alexa Fluor 555-labeled (red) donkey anti-mouse fluorescent secondary antibody (1:500; Beyotime, Shanghai, China) for an additional 1 h at 37 °C.

Techniques: Expressing, Infection, Quantitative RT-PCR, Control, Immunohistochemical staining, Staining

Journal: International Journal of Molecular Sciences

Article Title: UBE2G1 Is a Critical Component of Immune Response to the Infection of Pseudomonas Plecoglossicida in Large Yellow Croaker ( Larimichthys crocea )

doi: 10.3390/ijms23158298

Figure Lengend Snippet: SDS-PAGE analysis of recombinant Lc UBE2G1 fusion protein.

Article Snippet: The antibody incubation steps are as follows: firstly, the blocked cells were incubated with ubiquitin protein antibody (Mouse; 1:100; Abcam; Cambridge, MA, USA) and UBE2G1 protein antibody (Rabbit; 1:300; Irvine, Gentex Inc., Zeeland, MI, USA) at 37°C for 1 h; then incubated with Alexa Fluor 488-labeled (green) goat anti-rabbit, Alexa Fluor 555-labeled (red) donkey anti-mouse fluorescent secondary antibody (1:500; Beyotime, Shanghai, China) for an additional 1 h at 37 °C.

Techniques: SDS Page, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: UBE2G1 Is a Critical Component of Immune Response to the Infection of Pseudomonas Plecoglossicida in Large Yellow Croaker ( Larimichthys crocea )

doi: 10.3390/ijms23158298

Figure Lengend Snippet: Co-localization of Lc UBE2G1 and ubiquitin in Larimichthys crocea head kidney cells detected by laser confocal microscopy. ( A ) Lc UBE2G1 displayed by Alexa Fluor 488-labeled secondary antibody (green); ( B ) Ubiquitin (Ub) displayed by Alexa Fluor 555-labeled secondary antibody (red); ( C ) Cell nuclei stained with DAPI (blue); ( D ) The combined graphs of ( A – C ).

Article Snippet: The antibody incubation steps are as follows: firstly, the blocked cells were incubated with ubiquitin protein antibody (Mouse; 1:100; Abcam; Cambridge, MA, USA) and UBE2G1 protein antibody (Rabbit; 1:300; Irvine, Gentex Inc., Zeeland, MI, USA) at 37°C for 1 h; then incubated with Alexa Fluor 488-labeled (green) goat anti-rabbit, Alexa Fluor 555-labeled (red) donkey anti-mouse fluorescent secondary antibody (1:500; Beyotime, Shanghai, China) for an additional 1 h at 37 °C.

Techniques: Ubiquitin Proteomics, Confocal Microscopy, Labeling, Staining

Journal: International Journal of Molecular Sciences

Article Title: UBE2G1 Is a Critical Component of Immune Response to the Infection of Pseudomonas Plecoglossicida in Large Yellow Croaker ( Larimichthys crocea )

doi: 10.3390/ijms23158298

Figure Lengend Snippet: ( A ) SDS-PAGE analysis of GST pull-down results of recombinant Lc UBE2G1 fusion protein; ( B ) GST pull-down protein mass spectrometry results; ( C ) STRING analysis of proteins obtained by mass spectrometry.

Article Snippet: The antibody incubation steps are as follows: firstly, the blocked cells were incubated with ubiquitin protein antibody (Mouse; 1:100; Abcam; Cambridge, MA, USA) and UBE2G1 protein antibody (Rabbit; 1:300; Irvine, Gentex Inc., Zeeland, MI, USA) at 37°C for 1 h; then incubated with Alexa Fluor 488-labeled (green) goat anti-rabbit, Alexa Fluor 555-labeled (red) donkey anti-mouse fluorescent secondary antibody (1:500; Beyotime, Shanghai, China) for an additional 1 h at 37 °C.

Techniques: SDS Page, Recombinant, Mass Spectrometry

Journal: International Journal of Molecular Sciences

Article Title: UBE2G1 Is a Critical Component of Immune Response to the Infection of Pseudomonas Plecoglossicida in Large Yellow Croaker ( Larimichthys crocea )

doi: 10.3390/ijms23158298

Figure Lengend Snippet: Identity analysis of amino acid sequences of Ube2g1 in Larimichthys crocea and other species.

Article Snippet: The antibody incubation steps are as follows: firstly, the blocked cells were incubated with ubiquitin protein antibody (Mouse; 1:100; Abcam; Cambridge, MA, USA) and UBE2G1 protein antibody (Rabbit; 1:300; Irvine, Gentex Inc., Zeeland, MI, USA) at 37°C for 1 h; then incubated with Alexa Fluor 488-labeled (green) goat anti-rabbit, Alexa Fluor 555-labeled (red) donkey anti-mouse fluorescent secondary antibody (1:500; Beyotime, Shanghai, China) for an additional 1 h at 37 °C.

Techniques:

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet:

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

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

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/elife.40958

Figure Lengend Snippet: Figure 3. Loss of UBE2G1 blocked the degradation of cereblon neomorphic substrates induced by cereblon-modulating agents. (A and B) Immunoblot analysis of OPM2-Cas9 cells expressing non-targeting, UBE2G1-specific or CRBN-specific sgRNA. Cells were treated with LEN, POM or CC-220 for 16 hr (A) or CC-885 for 4 hr (B) at the indicated concentrations. (C) Immunoblot analysis of 293T parental or UBE2G1-/- cells treated with BRD4 PROTACs dBET1 or MZ1 at the indicated concentrations for 16 hr. (D) Immunoblot analysis of OPM2 parental or UBE2G1-/- cells treated with 100 mg/ml cycloheximide with or without 1 mM POM pretreatment for half an hour. Cells were harvested at the indicated time points. All results shown in this figure are representative of three independent experiments. DOI: https://doi.org/10.7554/eLife.40958.013 The following figure supplements are available for figure 3:

Article Snippet: Key resources table Reagent type Designation Source or reference Identifiers Additional information Cell line (human) U937 ATCC RRID:CVCL_0007 Cell line (human) OPM2 DSMZ RRID:CVCL_1625 Cell line (human) 293T Clontech Cat# 632180 Antibody anti-CRBN (rabbit monoclonal antibody) Celgene CRBN65 WB (1: 2,000) Antibody anti-UBE2G1 (mouse monoclonal antibody) Santa Cruz Biotechnology RRID:AB_1130981 WB (1: 500) Antibody anti-UBE2D3 (rabbit polyclonal antibody) Sigma-Aldrich RRID:AB_10605875 WB (1: 1,000) Antibody anti-GSPT1 (rabbit polyclonal antibody) Abcam RRID:AB_2115507 WB (1: 1,000) Antibody anti-IKZF1 (rabbit monoclonal antibody) Cell Signaling RRID:AB_2744523 WB (1: 1,000) Antibody anti-IKZF3 (rabbit monoclonal antibody) Cell Signaling RRID:AB_2744524 WB (1: 1,000) Antibody anti-CK1a(rabbit polyclonal antibody) Abcam RRID:AB_10864123 WB (1: 1,000) Antibody anti-ZFP91 (rabbit polyclonal antibody) LifeSpan Biosciences RRID:AB_2744522 WB (1: 1,000) Antibody anti-Cul4A (rabbit polyclonal antibody) Cell Signaling RRID:AB_2086563 WB (1: 1,000) Antibody anti-DDB1 (rabbit polyclonal antibody) Cell Signaling RRID:AB_10634753 WB (1: 1,000) Antibody anti-Rbx1 (rabbit polyclonal antibody) Cell Signaling RRID:AB_1904121 WB (1: 1,000) Antibody anti-HIS (mouse monoclonal antibody) Qiagen RRID:AB_2619735 WB (1: 1,000) Antibody anti-MBP (mouse monoclonal antibody) Santa Cruz Biotechnology RRID:AB_675707 WB (1: 1,000) Antibody anti-Actin (mouse monoclonal antibody) Sigma-Aldrich RRID:AB_476743 WB (1: 10,000) Antibody anti-Tubulin (mouse monoclonal antibody) Sigma-Aldrich RRID:AB_477593 WB (1: 10,000) Recombinant DNA reagent pcDNA3-8xHIS-Ub (plasmid) PMID:27338790 transient transfection Recombinant DNA reagent plenti-UBC-UBE2G1 -pGK-Pur (plasmid) this paper lentiviral transduction Recombinant DNA reagent plenti-UBC-UBE2G1 -C90S-pGK-Pur (plasmid) this paper lentiviral transduction Continued on next page Lu et al. eLife 2018;7:e40958.

Techniques: Western Blot, Expressing

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/elife.40958

Figure Lengend Snippet: Figure 7. UBE2G1-deficient OPM2 myeloma cells are resistant to lenalidomide and pomalidomide but remain sensitive to CC-220 at clinical relevant concentrations. (A and B) Cell proliferation (A) and immunoblot analysis (B) of OPM2-Cas9 cells transduced with lentiviral vectors expressing non- targeting, UBE2G1-specific or CRBN-specific sgRNAs. Cells were treated DMSO vehicle control, LEN, POM or CC-220 at the indicated concentrations for 5 days (A) or 16 hr (B). In (A), cell proliferation was determined by CTG, and data are presented as mean ± SD, n = 3 technical replicates. All results shown in this figure are representative of three independent experiments. DOI: https://doi.org/10.7554/eLife.40958.025 The following source data and figure supplements are available for figure 7:

Article Snippet: Key resources table Reagent type Designation Source or reference Identifiers Additional information Cell line (human) U937 ATCC RRID:CVCL_0007 Cell line (human) OPM2 DSMZ RRID:CVCL_1625 Cell line (human) 293T Clontech Cat# 632180 Antibody anti-CRBN (rabbit monoclonal antibody) Celgene CRBN65 WB (1: 2,000) Antibody anti-UBE2G1 (mouse monoclonal antibody) Santa Cruz Biotechnology RRID:AB_1130981 WB (1: 500) Antibody anti-UBE2D3 (rabbit polyclonal antibody) Sigma-Aldrich RRID:AB_10605875 WB (1: 1,000) Antibody anti-GSPT1 (rabbit polyclonal antibody) Abcam RRID:AB_2115507 WB (1: 1,000) Antibody anti-IKZF1 (rabbit monoclonal antibody) Cell Signaling RRID:AB_2744523 WB (1: 1,000) Antibody anti-IKZF3 (rabbit monoclonal antibody) Cell Signaling RRID:AB_2744524 WB (1: 1,000) Antibody anti-CK1a(rabbit polyclonal antibody) Abcam RRID:AB_10864123 WB (1: 1,000) Antibody anti-ZFP91 (rabbit polyclonal antibody) LifeSpan Biosciences RRID:AB_2744522 WB (1: 1,000) Antibody anti-Cul4A (rabbit polyclonal antibody) Cell Signaling RRID:AB_2086563 WB (1: 1,000) Antibody anti-DDB1 (rabbit polyclonal antibody) Cell Signaling RRID:AB_10634753 WB (1: 1,000) Antibody anti-Rbx1 (rabbit polyclonal antibody) Cell Signaling RRID:AB_1904121 WB (1: 1,000) Antibody anti-HIS (mouse monoclonal antibody) Qiagen RRID:AB_2619735 WB (1: 1,000) Antibody anti-MBP (mouse monoclonal antibody) Santa Cruz Biotechnology RRID:AB_675707 WB (1: 1,000) Antibody anti-Actin (mouse monoclonal antibody) Sigma-Aldrich RRID:AB_476743 WB (1: 10,000) Antibody anti-Tubulin (mouse monoclonal antibody) Sigma-Aldrich RRID:AB_477593 WB (1: 10,000) Recombinant DNA reagent pcDNA3-8xHIS-Ub (plasmid) PMID:27338790 transient transfection Recombinant DNA reagent plenti-UBC-UBE2G1 -pGK-Pur (plasmid) this paper lentiviral transduction Recombinant DNA reagent plenti-UBC-UBE2G1 -C90S-pGK-Pur (plasmid) this paper lentiviral transduction Continued on next page Lu et al. eLife 2018;7:e40958.

Techniques: Western Blot, Transduction, Expressing, Control