reference identifiers additional information antibody anti ube2g1 Search Results


ube2g1 rabbit polyclonal  (Bioss)
90
Bioss ube2g1 rabbit polyclonal
Ube2g1 Rabbit Polyclonal, supplied by Bioss, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ube2g1 rabbit polyclonal/product/Bioss
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anti ube2g1  (Proteintech)
91
Proteintech anti ube2g1

Anti Ube2g1, supplied by Proteintech, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti ube2i antibody  (Santa Cruz Biotechnology)
88
Santa Cruz Biotechnology anti ube2i antibody

Anti Ube2i Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit polyclonal anti ube2g1  (Boston Biochem)
86
Boston Biochem rabbit polyclonal anti ube2g1

Rabbit Polyclonal Anti Ube2g1, supplied by Boston Biochem, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti-ube2g1  (Millipore)
90
Millipore anti-ube2g1

Anti Ube2g1, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti-ube2g1/product/Millipore
Average 90 stars, based on 1 article reviews
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mouse anti-human ube2g1 mab  (Santa Cruz Biotechnology)
90
Santa Cruz Biotechnology mouse anti-human ube2g1 mab
(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 <t>UBE2G1-specific</t> sgRNAs. Cells were treated with POM at the indicated concentrations for 16 hours. Data are presented as mean ± SD (n = 4). (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 hours.
Mouse Anti Human Ube2g1 Mab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse anti-human ube2g1 mab/product/Santa Cruz Biotechnology
Average 90 stars, based on 1 article reviews
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anti-β-actin  (Millipore)
90
Millipore anti-β-actin
UBCH8, but not UBCH6 or UBCH9, is required for p21 degradation. (A) p21 in HCT116p53−/− cells is not degraded upon UV irradiation in the absence of UBCH8. Depletion of UBCH8 by two siRNA oligonucleotides (siUBCH8-A and siUBCH8-B) prevents UV-induced degradation of p21. Control cells were transfected with siRNA against luciferase (GL2). The Western blot shows the steady-state levels of p21 protein in UV-irradiated cells (+) and nonirradiated cells (−) as indicated. LC is a nonspecific band that cross-reacts with the anti-p21 antibody and is used as a loading control. (B) UBCH6 or UBCH9 mRNAs are decreased by their cognate siRNAs. The mRNA level of UBCH6 or UBCH9 was determined by quantitative real-time PCR, normalized to <t>β-actin</t> mRNA in the same sample and expressed as a percentage of the level in the si-GL2 sample. (C) UBCH8, but not UBE2G1 or UBE2G2, regulates p21 protein stability after UV irradiation. HCT116p53−/− cells were transfected with siRNA as indicated and treated with cycloheximide (CHX) for 10 min before UV irradiation. The Western blot shows the level of p21 protein at the indicated time points. Smaller quantities of protein lysates were loaded from the siUBCH8-A- or the si-UBE2G-transfected cells to ensure that the p21 signal at 0 min was comparable to that in the si-GL2-transfected cells (because si-UBCH8 increases the basal levels of p21; see text for details). The top blot shows a long exposure (exp.), and the middle blot shows a short exposure. Anti-β-actin was used as a loading control. (D) Quantitation of the p21 protein and the loading control signals was performed using GeneTools software. The plot shows the ratio of p21 to β-actin after normalizing the ratio at 0 min to 100.
Anti β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti-β-actin/product/Millipore
Average 90 stars, based on 1 article reviews
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anti-ubch8  (Proteintech)
90
Proteintech anti-ubch8
UBCH8, but not UBCH6 or UBCH9, is required for p21 degradation. (A) p21 in HCT116p53−/− cells is not degraded upon UV irradiation in the absence of UBCH8. Depletion of UBCH8 by two siRNA oligonucleotides (siUBCH8-A and siUBCH8-B) prevents UV-induced degradation of p21. Control cells were transfected with siRNA against luciferase (GL2). The Western blot shows the steady-state levels of p21 protein in UV-irradiated cells (+) and nonirradiated cells (−) as indicated. LC is a nonspecific band that cross-reacts with the anti-p21 antibody and is used as a loading control. (B) UBCH6 or UBCH9 mRNAs are decreased by their cognate siRNAs. The mRNA level of UBCH6 or UBCH9 was determined by quantitative real-time PCR, normalized to <t>β-actin</t> mRNA in the same sample and expressed as a percentage of the level in the si-GL2 sample. (C) UBCH8, but not UBE2G1 or UBE2G2, regulates p21 protein stability after UV irradiation. HCT116p53−/− cells were transfected with siRNA as indicated and treated with cycloheximide (CHX) for 10 min before UV irradiation. The Western blot shows the level of p21 protein at the indicated time points. Smaller quantities of protein lysates were loaded from the siUBCH8-A- or the si-UBE2G-transfected cells to ensure that the p21 signal at 0 min was comparable to that in the si-GL2-transfected cells (because si-UBCH8 increases the basal levels of p21; see text for details). The top blot shows a long exposure (exp.), and the middle blot shows a short exposure. Anti-β-actin was used as a loading control. (D) Quantitation of the p21 protein and the loading control signals was performed using GeneTools software. The plot shows the ratio of p21 to β-actin after normalizing the ratio at 0 min to 100.
Anti Ubch8, supplied by Proteintech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti-ubch8/product/Proteintech
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anti-α-actin  (Millipore)
90
Millipore anti-α-actin
UBCH8, but not UBCH6 or UBCH9, is required for p21 degradation. (A) p21 in HCT116p53−/− cells is not degraded upon UV irradiation in the absence of UBCH8. Depletion of UBCH8 by two siRNA oligonucleotides (siUBCH8-A and siUBCH8-B) prevents UV-induced degradation of p21. Control cells were transfected with siRNA against luciferase (GL2). The Western blot shows the steady-state levels of p21 protein in UV-irradiated cells (+) and nonirradiated cells (−) as indicated. LC is a nonspecific band that cross-reacts with the anti-p21 antibody and is used as a loading control. (B) UBCH6 or UBCH9 mRNAs are decreased by their cognate siRNAs. The mRNA level of UBCH6 or UBCH9 was determined by quantitative real-time PCR, normalized to <t>β-actin</t> mRNA in the same sample and expressed as a percentage of the level in the si-GL2 sample. (C) UBCH8, but not UBE2G1 or UBE2G2, regulates p21 protein stability after UV irradiation. HCT116p53−/− cells were transfected with siRNA as indicated and treated with cycloheximide (CHX) for 10 min before UV irradiation. The Western blot shows the level of p21 protein at the indicated time points. Smaller quantities of protein lysates were loaded from the siUBCH8-A- or the si-UBE2G-transfected cells to ensure that the p21 signal at 0 min was comparable to that in the si-GL2-transfected cells (because si-UBCH8 increases the basal levels of p21; see text for details). The top blot shows a long exposure (exp.), and the middle blot shows a short exposure. Anti-β-actin was used as a loading control. (D) Quantitation of the p21 protein and the loading control signals was performed using GeneTools software. The plot shows the ratio of p21 to β-actin after normalizing the ratio at 0 min to 100.
Anti α Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti-α-actin/product/Millipore
Average 90 stars, based on 1 article reviews
anti-α-actin - by Bioz Stars, 2026-03
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brd4 antibody 13440  (Cell Signaling Technology Inc)
90
Cell Signaling Technology Inc brd4 antibody 13440
UBCH8, but not UBCH6 or UBCH9, is required for p21 degradation. (A) p21 in HCT116p53−/− cells is not degraded upon UV irradiation in the absence of UBCH8. Depletion of UBCH8 by two siRNA oligonucleotides (siUBCH8-A and siUBCH8-B) prevents UV-induced degradation of p21. Control cells were transfected with siRNA against luciferase (GL2). The Western blot shows the steady-state levels of p21 protein in UV-irradiated cells (+) and nonirradiated cells (−) as indicated. LC is a nonspecific band that cross-reacts with the anti-p21 antibody and is used as a loading control. (B) UBCH6 or UBCH9 mRNAs are decreased by their cognate siRNAs. The mRNA level of UBCH6 or UBCH9 was determined by quantitative real-time PCR, normalized to <t>β-actin</t> mRNA in the same sample and expressed as a percentage of the level in the si-GL2 sample. (C) UBCH8, but not UBE2G1 or UBE2G2, regulates p21 protein stability after UV irradiation. HCT116p53−/− cells were transfected with siRNA as indicated and treated with cycloheximide (CHX) for 10 min before UV irradiation. The Western blot shows the level of p21 protein at the indicated time points. Smaller quantities of protein lysates were loaded from the siUBCH8-A- or the si-UBE2G-transfected cells to ensure that the p21 signal at 0 min was comparable to that in the si-GL2-transfected cells (because si-UBCH8 increases the basal levels of p21; see text for details). The top blot shows a long exposure (exp.), and the middle blot shows a short exposure. Anti-β-actin was used as a loading control. (D) Quantitation of the p21 protein and the loading control signals was performed using GeneTools software. The plot shows the ratio of p21 to β-actin after normalizing the ratio at 0 min to 100.
Brd4 Antibody 13440, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/brd4 antibody 13440/product/Cell Signaling Technology Inc
Average 90 stars, based on 1 article reviews
brd4 antibody 13440 - by Bioz Stars, 2026-03
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sarcomeric a-actinin  (Millipore)
90
Millipore sarcomeric a-actinin
UBCH8, but not UBCH6 or UBCH9, is required for p21 degradation. (A) p21 in HCT116p53−/− cells is not degraded upon UV irradiation in the absence of UBCH8. Depletion of UBCH8 by two siRNA oligonucleotides (siUBCH8-A and siUBCH8-B) prevents UV-induced degradation of p21. Control cells were transfected with siRNA against luciferase (GL2). The Western blot shows the steady-state levels of p21 protein in UV-irradiated cells (+) and nonirradiated cells (−) as indicated. LC is a nonspecific band that cross-reacts with the anti-p21 antibody and is used as a loading control. (B) UBCH6 or UBCH9 mRNAs are decreased by their cognate siRNAs. The mRNA level of UBCH6 or UBCH9 was determined by quantitative real-time PCR, normalized to <t>β-actin</t> mRNA in the same sample and expressed as a percentage of the level in the si-GL2 sample. (C) UBCH8, but not UBE2G1 or UBE2G2, regulates p21 protein stability after UV irradiation. HCT116p53−/− cells were transfected with siRNA as indicated and treated with cycloheximide (CHX) for 10 min before UV irradiation. The Western blot shows the level of p21 protein at the indicated time points. Smaller quantities of protein lysates were loaded from the siUBCH8-A- or the si-UBE2G-transfected cells to ensure that the p21 signal at 0 min was comparable to that in the si-GL2-transfected cells (because si-UBCH8 increases the basal levels of p21; see text for details). The top blot shows a long exposure (exp.), and the middle blot shows a short exposure. Anti-β-actin was used as a loading control. (D) Quantitation of the p21 protein and the loading control signals was performed using GeneTools software. The plot shows the ratio of p21 to β-actin after normalizing the ratio at 0 min to 100.
Sarcomeric A Actinin, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sarcomeric a-actinin/product/Millipore
Average 90 stars, based on 1 article reviews
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antibody anti-ikzf1 (rabbit monoclonal antibody)  (Cell Signaling Technology Inc)
90
Cell Signaling Technology Inc antibody anti-ikzf1 (rabbit monoclonal antibody)
UBCH8, but not UBCH6 or UBCH9, is required for p21 degradation. (A) p21 in HCT116p53−/− cells is not degraded upon UV irradiation in the absence of UBCH8. Depletion of UBCH8 by two siRNA oligonucleotides (siUBCH8-A and siUBCH8-B) prevents UV-induced degradation of p21. Control cells were transfected with siRNA against luciferase (GL2). The Western blot shows the steady-state levels of p21 protein in UV-irradiated cells (+) and nonirradiated cells (−) as indicated. LC is a nonspecific band that cross-reacts with the anti-p21 antibody and is used as a loading control. (B) UBCH6 or UBCH9 mRNAs are decreased by their cognate siRNAs. The mRNA level of UBCH6 or UBCH9 was determined by quantitative real-time PCR, normalized to <t>β-actin</t> mRNA in the same sample and expressed as a percentage of the level in the si-GL2 sample. (C) UBCH8, but not UBE2G1 or UBE2G2, regulates p21 protein stability after UV irradiation. HCT116p53−/− cells were transfected with siRNA as indicated and treated with cycloheximide (CHX) for 10 min before UV irradiation. The Western blot shows the level of p21 protein at the indicated time points. Smaller quantities of protein lysates were loaded from the siUBCH8-A- or the si-UBE2G-transfected cells to ensure that the p21 signal at 0 min was comparable to that in the si-GL2-transfected cells (because si-UBCH8 increases the basal levels of p21; see text for details). The top blot shows a long exposure (exp.), and the middle blot shows a short exposure. Anti-β-actin was used as a loading control. (D) Quantitation of the p21 protein and the loading control signals was performed using GeneTools software. The plot shows the ratio of p21 to β-actin after normalizing the ratio at 0 min to 100.
Antibody Anti Ikzf1 (Rabbit Monoclonal Antibody), supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/antibody anti-ikzf1 (rabbit monoclonal antibody)/product/Cell Signaling Technology Inc
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Image Search Results


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

(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 hours. Data are presented as mean ± SD (n = 4). (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 hours.

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

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 hours. Data are presented as mean ± SD (n = 4). (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 hours.

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam), rabbit anti-human IKZF1 mAb (Cell Signaling), rabbit anti-human IKZF3 mAb (Cell Signaling), rabbit anti-human CK1α pAb (Abcam), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences), mouse anti-human UBE2G1 mAb (Santa Cruz), rabbit anti-human UBE2D3 pAb (Sigma), rabbit anti-human Cul4A pAb (Cell Signaling), rabbit anti-human DDB1 pAb (Cell Signaling), rabbit anti-human Rbx1 pAb (Cell Signaling), rabbit anti-human Cdt1 pAb (Cell Signaling), rabbit anti-human Cdt2 pAb (Cell Signaling), rabbit anti-human Set8 pAb (Cell Signaling), rabbit anti-human RBM39 pAb (Sigma), rabbit anti-human p21 pAb (Cell Signaling), rabbit anti-human p27 pAb (Cell Signaling), rabbit anti-human c-Myc pAb (Cell Signaling), mouse anti-penta-HIS mAb (Qiagen), and mouse anti-human Actin and Tubulin antibodies (Sigma) were used as primary antibodies.

Techniques: CRISPR, Expressing, Western Blot, Mutagenesis

(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 hours. Data are presented as mean ± SD (n = 4). (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 hours. SE, short exposure; LE, long exposure.

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

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 hours. Data are presented as mean ± SD (n = 4). (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 hours. SE, short exposure; LE, long exposure.

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam), rabbit anti-human IKZF1 mAb (Cell Signaling), rabbit anti-human IKZF3 mAb (Cell Signaling), rabbit anti-human CK1α pAb (Abcam), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences), mouse anti-human UBE2G1 mAb (Santa Cruz), rabbit anti-human UBE2D3 pAb (Sigma), rabbit anti-human Cul4A pAb (Cell Signaling), rabbit anti-human DDB1 pAb (Cell Signaling), rabbit anti-human Rbx1 pAb (Cell Signaling), rabbit anti-human Cdt1 pAb (Cell Signaling), rabbit anti-human Cdt2 pAb (Cell Signaling), rabbit anti-human Set8 pAb (Cell Signaling), rabbit anti-human RBM39 pAb (Sigma), rabbit anti-human p21 pAb (Cell Signaling), rabbit anti-human p27 pAb (Cell Signaling), rabbit anti-human c-Myc pAb (Cell Signaling), mouse anti-penta-HIS mAb (Qiagen), and mouse anti-human Actin and Tubulin antibodies (Sigma) were used as primary antibodies.

Techniques: CRISPR, Expressing, Western Blot

The effect of double knockout of UBE2G1 and one of the 41 E2 enzymes on pomalidomide-induced destruction of IKZF1 (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 hours. Data are presented as mean ± SD (n = 4).

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: The effect of double knockout of UBE2G1 and one of the 41 E2 enzymes on pomalidomide-induced destruction of IKZF1 (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 hours. Data are presented as mean ± SD (n = 4).

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam), rabbit anti-human IKZF1 mAb (Cell Signaling), rabbit anti-human IKZF3 mAb (Cell Signaling), rabbit anti-human CK1α pAb (Abcam), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences), mouse anti-human UBE2G1 mAb (Santa Cruz), rabbit anti-human UBE2D3 pAb (Sigma), rabbit anti-human Cul4A pAb (Cell Signaling), rabbit anti-human DDB1 pAb (Cell Signaling), rabbit anti-human Rbx1 pAb (Cell Signaling), rabbit anti-human Cdt1 pAb (Cell Signaling), rabbit anti-human Cdt2 pAb (Cell Signaling), rabbit anti-human Set8 pAb (Cell Signaling), rabbit anti-human RBM39 pAb (Sigma), rabbit anti-human p21 pAb (Cell Signaling), rabbit anti-human p27 pAb (Cell Signaling), rabbit anti-human c-Myc pAb (Cell Signaling), mouse anti-penta-HIS mAb (Qiagen), and mouse anti-human Actin and Tubulin antibodies (Sigma) were used as primary antibodies.

Techniques: Double Knockout, Expressing

(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 hrs (A) or CC-885 for 4 hours (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 hours. (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.

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

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 hrs (A) or CC-885 for 4 hours (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 hours. (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.

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam), rabbit anti-human IKZF1 mAb (Cell Signaling), rabbit anti-human IKZF3 mAb (Cell Signaling), rabbit anti-human CK1α pAb (Abcam), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences), mouse anti-human UBE2G1 mAb (Santa Cruz), rabbit anti-human UBE2D3 pAb (Sigma), rabbit anti-human Cul4A pAb (Cell Signaling), rabbit anti-human DDB1 pAb (Cell Signaling), rabbit anti-human Rbx1 pAb (Cell Signaling), rabbit anti-human Cdt1 pAb (Cell Signaling), rabbit anti-human Cdt2 pAb (Cell Signaling), rabbit anti-human Set8 pAb (Cell Signaling), rabbit anti-human RBM39 pAb (Sigma), rabbit anti-human p21 pAb (Cell Signaling), rabbit anti-human p27 pAb (Cell Signaling), rabbit anti-human c-Myc pAb (Cell Signaling), mouse anti-penta-HIS mAb (Qiagen), and mouse anti-human Actin and Tubulin antibodies (Sigma) were used as primary antibodies.

Techniques: Western Blot, Expressing

Elimination of UBE2G1 blocks the degradation of cereblon neomorphic substrates recruited by lenalidomide and CC-885 (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 hours (A and B), or CC-885 for 4 hours (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 hours.

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: Elimination of UBE2G1 blocks the degradation of cereblon neomorphic substrates recruited by lenalidomide and CC-885 (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 hours (A and B), or CC-885 for 4 hours (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 hours.

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam), rabbit anti-human IKZF1 mAb (Cell Signaling), rabbit anti-human IKZF3 mAb (Cell Signaling), rabbit anti-human CK1α pAb (Abcam), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences), mouse anti-human UBE2G1 mAb (Santa Cruz), rabbit anti-human UBE2D3 pAb (Sigma), rabbit anti-human Cul4A pAb (Cell Signaling), rabbit anti-human DDB1 pAb (Cell Signaling), rabbit anti-human Rbx1 pAb (Cell Signaling), rabbit anti-human Cdt1 pAb (Cell Signaling), rabbit anti-human Cdt2 pAb (Cell Signaling), rabbit anti-human Set8 pAb (Cell Signaling), rabbit anti-human RBM39 pAb (Sigma), rabbit anti-human p21 pAb (Cell Signaling), rabbit anti-human p27 pAb (Cell Signaling), rabbit anti-human c-Myc pAb (Cell Signaling), mouse anti-penta-HIS mAb (Qiagen), and mouse anti-human Actin and Tubulin antibodies (Sigma) were used as primary antibodies.

Techniques: Western Blot, Transduction, Expressing, Stable Transfection, Mutagenesis

(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 hours (B). In (A), cell proliferation was determined by CTG, and data are presented as mean ± SD (n=3).

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

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 hours (B). In (A), cell proliferation was determined by CTG, and data are presented as mean ± SD (n=3).

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam), rabbit anti-human IKZF1 mAb (Cell Signaling), rabbit anti-human IKZF3 mAb (Cell Signaling), rabbit anti-human CK1α pAb (Abcam), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences), mouse anti-human UBE2G1 mAb (Santa Cruz), rabbit anti-human UBE2D3 pAb (Sigma), rabbit anti-human Cul4A pAb (Cell Signaling), rabbit anti-human DDB1 pAb (Cell Signaling), rabbit anti-human Rbx1 pAb (Cell Signaling), rabbit anti-human Cdt1 pAb (Cell Signaling), rabbit anti-human Cdt2 pAb (Cell Signaling), rabbit anti-human Set8 pAb (Cell Signaling), rabbit anti-human RBM39 pAb (Sigma), rabbit anti-human p21 pAb (Cell Signaling), rabbit anti-human p27 pAb (Cell Signaling), rabbit anti-human c-Myc pAb (Cell Signaling), mouse anti-penta-HIS mAb (Qiagen), and mouse anti-human Actin and Tubulin antibodies (Sigma) were used as primary antibodies.

Techniques: Western Blot, Transduction, Expressing

UBE2G1 knockout diminished the responses to lenalidomide, pomalidomide and CC-220 in myeloma cell lines DF15 and MM1S (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 hours (B and D). In (A and C), cell proliferation was determined by CTG, and data are presented as mean ± SD (n=3).

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: UBE2G1 knockout diminished the responses to lenalidomide, pomalidomide and CC-220 in myeloma cell lines DF15 and MM1S (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 hours (B and D). In (A and C), cell proliferation was determined by CTG, and data are presented as mean ± SD (n=3).

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam), rabbit anti-human IKZF1 mAb (Cell Signaling), rabbit anti-human IKZF3 mAb (Cell Signaling), rabbit anti-human CK1α pAb (Abcam), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences), mouse anti-human UBE2G1 mAb (Santa Cruz), rabbit anti-human UBE2D3 pAb (Sigma), rabbit anti-human Cul4A pAb (Cell Signaling), rabbit anti-human DDB1 pAb (Cell Signaling), rabbit anti-human Rbx1 pAb (Cell Signaling), rabbit anti-human Cdt1 pAb (Cell Signaling), rabbit anti-human Cdt2 pAb (Cell Signaling), rabbit anti-human Set8 pAb (Cell Signaling), rabbit anti-human RBM39 pAb (Sigma), rabbit anti-human p21 pAb (Cell Signaling), rabbit anti-human p27 pAb (Cell Signaling), rabbit anti-human c-Myc pAb (Cell Signaling), mouse anti-penta-HIS mAb (Qiagen), and mouse anti-human Actin and Tubulin antibodies (Sigma) were used as primary antibodies.

Techniques: Knock-Out, Western Blot, Infection, Expressing

UBE2G1 catalyzes the ubiquitin chain assembly on GSPT1 pre-conjugated with ubiquitin (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 hours. 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.

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: UBE2G1 catalyzes the ubiquitin chain assembly on GSPT1 pre-conjugated with ubiquitin (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 hours. 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.

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam), rabbit anti-human IKZF1 mAb (Cell Signaling), rabbit anti-human IKZF3 mAb (Cell Signaling), rabbit anti-human CK1α pAb (Abcam), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences), mouse anti-human UBE2G1 mAb (Santa Cruz), rabbit anti-human UBE2D3 pAb (Sigma), rabbit anti-human Cul4A pAb (Cell Signaling), rabbit anti-human DDB1 pAb (Cell Signaling), rabbit anti-human Rbx1 pAb (Cell Signaling), rabbit anti-human Cdt1 pAb (Cell Signaling), rabbit anti-human Cdt2 pAb (Cell Signaling), rabbit anti-human Set8 pAb (Cell Signaling), rabbit anti-human RBM39 pAb (Sigma), rabbit anti-human p21 pAb (Cell Signaling), rabbit anti-human p27 pAb (Cell Signaling), rabbit anti-human c-Myc pAb (Cell Signaling), mouse anti-penta-HIS mAb (Qiagen), and mouse anti-human Actin and Tubulin antibodies (Sigma) were used as primary antibodies.

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

(A-D) In vitro ubiquitination of IKZF1 (A and C) and GSPT1 (B and D) MBP fusion proteins by recombinant CRL4CRBN 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 hours, and then analyzed by immunoblotting. (E) Sequential in vitro ubiquitination of GSPT1 by recombinant CRL4CRBN 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 hours. 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 hours, followed by immunoblot analysis. (F) Schematic showing the sequential ubiquitination of CRBN neomorphic substrates by UBE2D3 and UBE2G1.

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: (A-D) In vitro ubiquitination of IKZF1 (A and C) and GSPT1 (B and D) MBP fusion proteins by recombinant CRL4CRBN 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 hours, and then analyzed by immunoblotting. (E) Sequential in vitro ubiquitination of GSPT1 by recombinant CRL4CRBN 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 hours. 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 hours, followed by immunoblot analysis. (F) Schematic showing the sequential ubiquitination of CRBN neomorphic substrates by UBE2D3 and UBE2G1.

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam), rabbit anti-human IKZF1 mAb (Cell Signaling), rabbit anti-human IKZF3 mAb (Cell Signaling), rabbit anti-human CK1α pAb (Abcam), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences), mouse anti-human UBE2G1 mAb (Santa Cruz), rabbit anti-human UBE2D3 pAb (Sigma), rabbit anti-human Cul4A pAb (Cell Signaling), rabbit anti-human DDB1 pAb (Cell Signaling), rabbit anti-human Rbx1 pAb (Cell Signaling), rabbit anti-human Cdt1 pAb (Cell Signaling), rabbit anti-human Cdt2 pAb (Cell Signaling), rabbit anti-human Set8 pAb (Cell Signaling), rabbit anti-human RBM39 pAb (Sigma), rabbit anti-human p21 pAb (Cell Signaling), rabbit anti-human p27 pAb (Cell Signaling), rabbit anti-human c-Myc pAb (Cell Signaling), mouse anti-penta-HIS mAb (Qiagen), and mouse anti-human Actin and Tubulin antibodies (Sigma) were used as primary antibodies.

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

(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 transfect with plasmids expressing cereblon, IKZF1-V5, 8xHis-Ub with or without UBE2G1 wild-type or C90S mutant. In (A), (B) and (C), 48 hours after transfection, cells were treated with MG-132 (10 μM) and POM at the indicated concentrations for additional 8 hours. 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 .

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

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 transfect with plasmids expressing cereblon, IKZF1-V5, 8xHis-Ub with or without UBE2G1 wild-type or C90S mutant. In (A), (B) and (C), 48 hours after transfection, cells were treated with MG-132 (10 μM) and POM at the indicated concentrations for additional 8 hours. 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 .

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam), rabbit anti-human IKZF1 mAb (Cell Signaling), rabbit anti-human IKZF3 mAb (Cell Signaling), rabbit anti-human CK1α pAb (Abcam), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences), mouse anti-human UBE2G1 mAb (Santa Cruz), rabbit anti-human UBE2D3 pAb (Sigma), rabbit anti-human Cul4A pAb (Cell Signaling), rabbit anti-human DDB1 pAb (Cell Signaling), rabbit anti-human Rbx1 pAb (Cell Signaling), rabbit anti-human Cdt1 pAb (Cell Signaling), rabbit anti-human Cdt2 pAb (Cell Signaling), rabbit anti-human Set8 pAb (Cell Signaling), rabbit anti-human RBM39 pAb (Sigma), rabbit anti-human p21 pAb (Cell Signaling), rabbit anti-human p27 pAb (Cell Signaling), rabbit anti-human c-Myc pAb (Cell Signaling), mouse anti-penta-HIS mAb (Qiagen), and mouse anti-human Actin and Tubulin antibodies (Sigma) were used as primary antibodies.

Techniques: Transfection, Expressing, Mutagenesis, Western Blot

Input protein levels for the in vivo ubiquitinaiton studies corresponding to (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 hours after transfection, cells were treated with 10 μM MG132 and POM at the indicated concentrations for additional 8 hours.

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: Input protein levels for the in vivo ubiquitinaiton studies corresponding to (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 hours after transfection, cells were treated with 10 μM MG132 and POM at the indicated concentrations for additional 8 hours.

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam), rabbit anti-human IKZF1 mAb (Cell Signaling), rabbit anti-human IKZF3 mAb (Cell Signaling), rabbit anti-human CK1α pAb (Abcam), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences), mouse anti-human UBE2G1 mAb (Santa Cruz), rabbit anti-human UBE2D3 pAb (Sigma), rabbit anti-human Cul4A pAb (Cell Signaling), rabbit anti-human DDB1 pAb (Cell Signaling), rabbit anti-human Rbx1 pAb (Cell Signaling), rabbit anti-human Cdt1 pAb (Cell Signaling), rabbit anti-human Cdt2 pAb (Cell Signaling), rabbit anti-human Set8 pAb (Cell Signaling), rabbit anti-human RBM39 pAb (Sigma), rabbit anti-human p21 pAb (Cell Signaling), rabbit anti-human p27 pAb (Cell Signaling), rabbit anti-human c-Myc pAb (Cell Signaling), mouse anti-penta-HIS mAb (Qiagen), and mouse anti-human Actin and Tubulin antibodies (Sigma) were used as primary antibodies.

Techniques: In Vivo, Western Blot, Transfection, Mutagenesis

(A) Effect of lenalidomide (top panel) and pomalidomide (bottom panel) on proliferation of myeloma cell lines. Cell proliferation was determined by CTG. Data are presented as mean ± SD (n=3). (B) Immunoblot analysis of myeloma cell lines. (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 hours (D). In (C), cell proliferation was determined by CTG, and data are presented as mean ± SD (n=3).

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: (A) Effect of lenalidomide (top panel) and pomalidomide (bottom panel) on proliferation of myeloma cell lines. Cell proliferation was determined by CTG. Data are presented as mean ± SD (n=3). (B) Immunoblot analysis of myeloma cell lines. (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 hours (D). In (C), cell proliferation was determined by CTG, and data are presented as mean ± SD (n=3).

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam), rabbit anti-human IKZF1 mAb (Cell Signaling), rabbit anti-human IKZF3 mAb (Cell Signaling), rabbit anti-human CK1α pAb (Abcam), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences), mouse anti-human UBE2G1 mAb (Santa Cruz), rabbit anti-human UBE2D3 pAb (Sigma), rabbit anti-human Cul4A pAb (Cell Signaling), rabbit anti-human DDB1 pAb (Cell Signaling), rabbit anti-human Rbx1 pAb (Cell Signaling), rabbit anti-human Cdt1 pAb (Cell Signaling), rabbit anti-human Cdt2 pAb (Cell Signaling), rabbit anti-human Set8 pAb (Cell Signaling), rabbit anti-human RBM39 pAb (Sigma), rabbit anti-human p21 pAb (Cell Signaling), rabbit anti-human p27 pAb (Cell Signaling), rabbit anti-human c-Myc pAb (Cell Signaling), mouse anti-penta-HIS mAb (Qiagen), and mouse anti-human Actin and Tubulin antibodies (Sigma) were used as primary antibodies.

Techniques: Western Blot, Transduction

The growth-inhibitory effect of CC-885 and Brd4 PROTACs in AML cell lines and 293T cells (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 hours. Cell proliferation was determined by CTG. Data are presented as mean ± SD (n=4). (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).

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: The growth-inhibitory effect of CC-885 and Brd4 PROTACs in AML cell lines and 293T cells (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 hours. Cell proliferation was determined by CTG. Data are presented as mean ± SD (n=4). (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).

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam), rabbit anti-human IKZF1 mAb (Cell Signaling), rabbit anti-human IKZF3 mAb (Cell Signaling), rabbit anti-human CK1α pAb (Abcam), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences), mouse anti-human UBE2G1 mAb (Santa Cruz), rabbit anti-human UBE2D3 pAb (Sigma), rabbit anti-human Cul4A pAb (Cell Signaling), rabbit anti-human DDB1 pAb (Cell Signaling), rabbit anti-human Rbx1 pAb (Cell Signaling), rabbit anti-human Cdt1 pAb (Cell Signaling), rabbit anti-human Cdt2 pAb (Cell Signaling), rabbit anti-human Set8 pAb (Cell Signaling), rabbit anti-human RBM39 pAb (Sigma), rabbit anti-human p21 pAb (Cell Signaling), rabbit anti-human p27 pAb (Cell Signaling), rabbit anti-human c-Myc pAb (Cell Signaling), mouse anti-penta-HIS mAb (Qiagen), and mouse anti-human Actin and Tubulin antibodies (Sigma) were used as primary antibodies.

Techniques: Over Expression, Mutagenesis

Depletion of UBE2G1 attenuated the degradation of p21 and RMB39 induced by UV irradiation and sulfonamide treatment, respectively. (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 hours.

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: Depletion of UBE2G1 attenuated the degradation of p21 and RMB39 induced by UV irradiation and sulfonamide treatment, respectively. (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 hours.

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam), rabbit anti-human IKZF1 mAb (Cell Signaling), rabbit anti-human IKZF3 mAb (Cell Signaling), rabbit anti-human CK1α pAb (Abcam), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences), mouse anti-human UBE2G1 mAb (Santa Cruz), rabbit anti-human UBE2D3 pAb (Sigma), rabbit anti-human Cul4A pAb (Cell Signaling), rabbit anti-human DDB1 pAb (Cell Signaling), rabbit anti-human Rbx1 pAb (Cell Signaling), rabbit anti-human Cdt1 pAb (Cell Signaling), rabbit anti-human Cdt2 pAb (Cell Signaling), rabbit anti-human Set8 pAb (Cell Signaling), rabbit anti-human RBM39 pAb (Sigma), rabbit anti-human p21 pAb (Cell Signaling), rabbit anti-human p27 pAb (Cell Signaling), rabbit anti-human c-Myc pAb (Cell Signaling), mouse anti-penta-HIS mAb (Qiagen), and mouse anti-human Actin and Tubulin antibodies (Sigma) were used as primary antibodies.

Techniques: Irradiation, Western Blot, Concentration Assay

UBE2D family proteins redundantly promote the ubiquitination of GSPT1 (A) Sequence alignment of human UBE2D family proteins using Clustal W 2.1. Note that the amino acid sequence identity among all 4 family proteins is close to 90%. (B) In vitro ubiquitination of GSPT1 MBP fusion protein by recombinant CRL4CRBN 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 hours, and then analyzed by immunoblotting. SE, short exposure; LE, long exposure

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: UBE2D family proteins redundantly promote the ubiquitination of GSPT1 (A) Sequence alignment of human UBE2D family proteins using Clustal W 2.1. Note that the amino acid sequence identity among all 4 family proteins is close to 90%. (B) In vitro ubiquitination of GSPT1 MBP fusion protein by recombinant CRL4CRBN 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 hours, and then analyzed by immunoblotting. SE, short exposure; LE, long exposure

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam), rabbit anti-human IKZF1 mAb (Cell Signaling), rabbit anti-human IKZF3 mAb (Cell Signaling), rabbit anti-human CK1α pAb (Abcam), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences), mouse anti-human UBE2G1 mAb (Santa Cruz), rabbit anti-human UBE2D3 pAb (Sigma), rabbit anti-human Cul4A pAb (Cell Signaling), rabbit anti-human DDB1 pAb (Cell Signaling), rabbit anti-human Rbx1 pAb (Cell Signaling), rabbit anti-human Cdt1 pAb (Cell Signaling), rabbit anti-human Cdt2 pAb (Cell Signaling), rabbit anti-human Set8 pAb (Cell Signaling), rabbit anti-human RBM39 pAb (Sigma), rabbit anti-human p21 pAb (Cell Signaling), rabbit anti-human p27 pAb (Cell Signaling), rabbit anti-human c-Myc pAb (Cell Signaling), mouse anti-penta-HIS mAb (Qiagen), and mouse anti-human Actin and Tubulin antibodies (Sigma) were used as primary antibodies.

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

UBCH8, but not UBCH6 or UBCH9, is required for p21 degradation. (A) p21 in HCT116p53−/− cells is not degraded upon UV irradiation in the absence of UBCH8. Depletion of UBCH8 by two siRNA oligonucleotides (siUBCH8-A and siUBCH8-B) prevents UV-induced degradation of p21. Control cells were transfected with siRNA against luciferase (GL2). The Western blot shows the steady-state levels of p21 protein in UV-irradiated cells (+) and nonirradiated cells (−) as indicated. LC is a nonspecific band that cross-reacts with the anti-p21 antibody and is used as a loading control. (B) UBCH6 or UBCH9 mRNAs are decreased by their cognate siRNAs. The mRNA level of UBCH6 or UBCH9 was determined by quantitative real-time PCR, normalized to β-actin mRNA in the same sample and expressed as a percentage of the level in the si-GL2 sample. (C) UBCH8, but not UBE2G1 or UBE2G2, regulates p21 protein stability after UV irradiation. HCT116p53−/− cells were transfected with siRNA as indicated and treated with cycloheximide (CHX) for 10 min before UV irradiation. The Western blot shows the level of p21 protein at the indicated time points. Smaller quantities of protein lysates were loaded from the siUBCH8-A- or the si-UBE2G-transfected cells to ensure that the p21 signal at 0 min was comparable to that in the si-GL2-transfected cells (because si-UBCH8 increases the basal levels of p21; see text for details). The top blot shows a long exposure (exp.), and the middle blot shows a short exposure. Anti-β-actin was used as a loading control. (D) Quantitation of the p21 protein and the loading control signals was performed using GeneTools software. The plot shows the ratio of p21 to β-actin after normalizing the ratio at 0 min to 100.

Journal: Molecular and Cellular Biology

Article Title: Selective Ubiquitylation of p21 and Cdt1 by UBCH8 and UBE2G Ubiquitin-Conjugating Enzymes via the CRL4 Cdt2 Ubiquitin Ligase Complex ▿ †

doi: 10.1128/MCB.05496-11

Figure Lengend Snippet: UBCH8, but not UBCH6 or UBCH9, is required for p21 degradation. (A) p21 in HCT116p53−/− cells is not degraded upon UV irradiation in the absence of UBCH8. Depletion of UBCH8 by two siRNA oligonucleotides (siUBCH8-A and siUBCH8-B) prevents UV-induced degradation of p21. Control cells were transfected with siRNA against luciferase (GL2). The Western blot shows the steady-state levels of p21 protein in UV-irradiated cells (+) and nonirradiated cells (−) as indicated. LC is a nonspecific band that cross-reacts with the anti-p21 antibody and is used as a loading control. (B) UBCH6 or UBCH9 mRNAs are decreased by their cognate siRNAs. The mRNA level of UBCH6 or UBCH9 was determined by quantitative real-time PCR, normalized to β-actin mRNA in the same sample and expressed as a percentage of the level in the si-GL2 sample. (C) UBCH8, but not UBE2G1 or UBE2G2, regulates p21 protein stability after UV irradiation. HCT116p53−/− cells were transfected with siRNA as indicated and treated with cycloheximide (CHX) for 10 min before UV irradiation. The Western blot shows the level of p21 protein at the indicated time points. Smaller quantities of protein lysates were loaded from the siUBCH8-A- or the si-UBE2G-transfected cells to ensure that the p21 signal at 0 min was comparable to that in the si-GL2-transfected cells (because si-UBCH8 increases the basal levels of p21; see text for details). The top blot shows a long exposure (exp.), and the middle blot shows a short exposure. Anti-β-actin was used as a loading control. (D) Quantitation of the p21 protein and the loading control signals was performed using GeneTools software. The plot shows the ratio of p21 to β-actin after normalizing the ratio at 0 min to 100.

Article Snippet: Anti-p21 (C-19; Santa Cruz Biotechnology), anti-UBCH8 (Proteintech Group, Inc.), anti-UBE2G1 (Sigma), anti-β-actin (Sigma), and anti-tubulin (Santa Cruz Biotechnology) antibodies were purchased from the indicated companies.

Techniques: Irradiation, Transfection, Luciferase, Western Blot, Real-time Polymerase Chain Reaction, Quantitation Assay, Software

UBCH8 destabilizes p21 during the normal cell cycle. (A) The half-life of p21 protein is increased by knockdown of UBCH8. siRNA-transfected cells were treated with cycloheximide (CHX) for the indicated time. The level of p21 was analyzed by Western blotting. Smaller quantities of protein lysates were loaded from the si-UBCH8-A-transfected cells to ensure that the signal at 0 min was comparable to that in the si-GL2-transfected cells. (B) Quantitation of the p21 protein and the loading control signals was performed using GeneTools software. The ratio of p21 to β-actin was plotted after normalizing the ratio at 0 h to 100. (C) p21 mRNA is not affected by knockdown of UBCH8. The p21 mRNA levels in siRNA-transfected cells were determined by quantitative real-time PCR, normalized to β-actin, and the ratio was expressed relative to the control si-GL2 sample. (D) UBCH8 overexpression destabilizes p21 protein. The p21 level in Myc-UBCH8-overexpressing HCT116p53−/− cells was determined by Western blotting. Three independent clones of cells were analyzed. β-Actin is used as loading control. (E) Quantitation of the p21 protein and the loading control signals was performed using GeneTools software. The ratio of p21 to β-actin was plotted. (F) The basal level of p21 mRNA is not significantly decreased by overexpression of Myc-UBCH8 as determined by quantitative real-time PCR in the same clones of cells as shown in panel B. The mRNA level of p21 was normalized to β-actin and expressed relative to the ratio in vector-transfected control cells. (G) Vector-control or Myc-UBCH8-expressing HCT116p53−/− cells were transfected with siRNA against the open reading frame (ORF) (si-UBCH8-A) or the 3′UTR (si-UBCH8-B) of UBCH8. Immunoblots are shown with the indicated antibodies. The asterisk indicates a degradation product of overexpressed UBCH8. For the UBCH8 blot, a lower exposure is shown for lanes 4 to 6 to prevent saturation of signal. β-Actin is shown to control for protein loading.

Journal: Molecular and Cellular Biology

Article Title: Selective Ubiquitylation of p21 and Cdt1 by UBCH8 and UBE2G Ubiquitin-Conjugating Enzymes via the CRL4 Cdt2 Ubiquitin Ligase Complex ▿ †

doi: 10.1128/MCB.05496-11

Figure Lengend Snippet: UBCH8 destabilizes p21 during the normal cell cycle. (A) The half-life of p21 protein is increased by knockdown of UBCH8. siRNA-transfected cells were treated with cycloheximide (CHX) for the indicated time. The level of p21 was analyzed by Western blotting. Smaller quantities of protein lysates were loaded from the si-UBCH8-A-transfected cells to ensure that the signal at 0 min was comparable to that in the si-GL2-transfected cells. (B) Quantitation of the p21 protein and the loading control signals was performed using GeneTools software. The ratio of p21 to β-actin was plotted after normalizing the ratio at 0 h to 100. (C) p21 mRNA is not affected by knockdown of UBCH8. The p21 mRNA levels in siRNA-transfected cells were determined by quantitative real-time PCR, normalized to β-actin, and the ratio was expressed relative to the control si-GL2 sample. (D) UBCH8 overexpression destabilizes p21 protein. The p21 level in Myc-UBCH8-overexpressing HCT116p53−/− cells was determined by Western blotting. Three independent clones of cells were analyzed. β-Actin is used as loading control. (E) Quantitation of the p21 protein and the loading control signals was performed using GeneTools software. The ratio of p21 to β-actin was plotted. (F) The basal level of p21 mRNA is not significantly decreased by overexpression of Myc-UBCH8 as determined by quantitative real-time PCR in the same clones of cells as shown in panel B. The mRNA level of p21 was normalized to β-actin and expressed relative to the ratio in vector-transfected control cells. (G) Vector-control or Myc-UBCH8-expressing HCT116p53−/− cells were transfected with siRNA against the open reading frame (ORF) (si-UBCH8-A) or the 3′UTR (si-UBCH8-B) of UBCH8. Immunoblots are shown with the indicated antibodies. The asterisk indicates a degradation product of overexpressed UBCH8. For the UBCH8 blot, a lower exposure is shown for lanes 4 to 6 to prevent saturation of signal. β-Actin is shown to control for protein loading.

Article Snippet: Anti-p21 (C-19; Santa Cruz Biotechnology), anti-UBCH8 (Proteintech Group, Inc.), anti-UBE2G1 (Sigma), anti-β-actin (Sigma), and anti-tubulin (Santa Cruz Biotechnology) antibodies were purchased from the indicated companies.

Techniques: Transfection, Western Blot, Quantitation Assay, Software, Real-time Polymerase Chain Reaction, Over Expression, Clone Assay, Plasmid Preparation, Expressing

UBCH6, UBCH8, and UBCH9 are not required for UV-induced Cdt1 degradation. (A and B) The degradation of Cdt1 after UV irradiation in HCT116p53−/− cells depleted of UBCH6, UBCH8, or UBCH9 (UBCH6/8/9)is not impaired. Western blot analysis of Cdt1 steady-state level following UV irradiation in cells depleted of the indicated ubiquitin-conjugating enzymes either individually (A) or in combination (B) by siRNA. Cells transfected with siRNA against luciferase (GL2) are used as a control. The results demonstrate that UBCH6, UBCH8, and UBCH9 are not required for efficient degradation of Cdt1 postradiation. (C) Quantitation of the Cdt1 protein and β-actin shown in panel B was performed using GeneTools software. The ratio of Cdt1 to β-actin was plotted after normalizing the ratio at 0 min to 100.

Journal: Molecular and Cellular Biology

Article Title: Selective Ubiquitylation of p21 and Cdt1 by UBCH8 and UBE2G Ubiquitin-Conjugating Enzymes via the CRL4 Cdt2 Ubiquitin Ligase Complex ▿ †

doi: 10.1128/MCB.05496-11

Figure Lengend Snippet: UBCH6, UBCH8, and UBCH9 are not required for UV-induced Cdt1 degradation. (A and B) The degradation of Cdt1 after UV irradiation in HCT116p53−/− cells depleted of UBCH6, UBCH8, or UBCH9 (UBCH6/8/9)is not impaired. Western blot analysis of Cdt1 steady-state level following UV irradiation in cells depleted of the indicated ubiquitin-conjugating enzymes either individually (A) or in combination (B) by siRNA. Cells transfected with siRNA against luciferase (GL2) are used as a control. The results demonstrate that UBCH6, UBCH8, and UBCH9 are not required for efficient degradation of Cdt1 postradiation. (C) Quantitation of the Cdt1 protein and β-actin shown in panel B was performed using GeneTools software. The ratio of Cdt1 to β-actin was plotted after normalizing the ratio at 0 min to 100.

Article Snippet: Anti-p21 (C-19; Santa Cruz Biotechnology), anti-UBCH8 (Proteintech Group, Inc.), anti-UBE2G1 (Sigma), anti-β-actin (Sigma), and anti-tubulin (Santa Cruz Biotechnology) antibodies were purchased from the indicated companies.

Techniques: Irradiation, Western Blot, Transfection, Luciferase, Quantitation Assay, Software

UBE2G1 and UBE2G2 are required for UV-induced Cdt1 degradation. (A) HCT116p53−/− cells depleted of UBE2G1 or UBE2G2 are deficient in their ability to degrade Cdt1 postradiation. The results of Western blot analysis of the Cdt1 steady-state level following UV irradiation in cells depleted of the indicated ubiquitin-conjugating enzymes by siRNA are shown. The degree of Cdt1 degradation postradiation was determined by quantitating the signal intensity of Cdt1 (normalized to β-actin) and expressed as a percentage of that in unirradiated cells of the corresponding siRNA transfection. (B) Efficient knockdown of UBE2G1 or UBE2G2 by the cognate siRNAs. HCT116p53−/− cells were transfected with siRNA targeting the indicated proteins for 72 h before harvesting. (Left) The protein level of UBE2G1 was determined by Western blotting. The lowest band is the specific band. (Right) The mRNA level of UBE2G2 was determined by quantitative real-time PCR, normalized to that of β-actin and expressed relative to that of the control si-GL2 sample. (C) Coknockdown of UBE2G1 and UBE2G2 by two separate siRNAs (-A or -B for each gene) prevents the degradation of Cdt1 after UV irradiation. The levels were measured as described above for panel A. The extent of Cdt1 degradation postradiation was determined as described above for panel A. (D) Cdt1 mRNA is not increased by knockdown of UBE2G1 and UBE2G2. The levels were measured as described above for panel B. (E) The half-life of Cdt1 protein after UV irradiation is increased by knockdown of UBE2G1 and UBE2G2. (F) Quantitation of the Cdt1 protein and β-actin was performed using GeneTools software. The ratio of Cdt1 to β-actin was plotted after normalizing the ratio at 0 min to 100.

Journal: Molecular and Cellular Biology

Article Title: Selective Ubiquitylation of p21 and Cdt1 by UBCH8 and UBE2G Ubiquitin-Conjugating Enzymes via the CRL4 Cdt2 Ubiquitin Ligase Complex ▿ †

doi: 10.1128/MCB.05496-11

Figure Lengend Snippet: UBE2G1 and UBE2G2 are required for UV-induced Cdt1 degradation. (A) HCT116p53−/− cells depleted of UBE2G1 or UBE2G2 are deficient in their ability to degrade Cdt1 postradiation. The results of Western blot analysis of the Cdt1 steady-state level following UV irradiation in cells depleted of the indicated ubiquitin-conjugating enzymes by siRNA are shown. The degree of Cdt1 degradation postradiation was determined by quantitating the signal intensity of Cdt1 (normalized to β-actin) and expressed as a percentage of that in unirradiated cells of the corresponding siRNA transfection. (B) Efficient knockdown of UBE2G1 or UBE2G2 by the cognate siRNAs. HCT116p53−/− cells were transfected with siRNA targeting the indicated proteins for 72 h before harvesting. (Left) The protein level of UBE2G1 was determined by Western blotting. The lowest band is the specific band. (Right) The mRNA level of UBE2G2 was determined by quantitative real-time PCR, normalized to that of β-actin and expressed relative to that of the control si-GL2 sample. (C) Coknockdown of UBE2G1 and UBE2G2 by two separate siRNAs (-A or -B for each gene) prevents the degradation of Cdt1 after UV irradiation. The levels were measured as described above for panel A. The extent of Cdt1 degradation postradiation was determined as described above for panel A. (D) Cdt1 mRNA is not increased by knockdown of UBE2G1 and UBE2G2. The levels were measured as described above for panel B. (E) The half-life of Cdt1 protein after UV irradiation is increased by knockdown of UBE2G1 and UBE2G2. (F) Quantitation of the Cdt1 protein and β-actin was performed using GeneTools software. The ratio of Cdt1 to β-actin was plotted after normalizing the ratio at 0 min to 100.

Article Snippet: Anti-p21 (C-19; Santa Cruz Biotechnology), anti-UBCH8 (Proteintech Group, Inc.), anti-UBE2G1 (Sigma), anti-β-actin (Sigma), and anti-tubulin (Santa Cruz Biotechnology) antibodies were purchased from the indicated companies.

Techniques: Western Blot, Irradiation, Transfection, Real-time Polymerase Chain Reaction, Quantitation Assay, Software

UBE2G1 and UBE2G2 regulate p21 transcriptionally but do not affect its protein stability. (A) Basal level of p21 protein increased after knockdown of UBE2G1 or UBE2G2. The level was measured as described in the legend of Fig. 1A. (B) p21 mRNA induced by knockdown of UBE2G1 or UBE2G2. The p21 mRNA level in siRNA-transfected cells was determined by quantitative real-time PCR, normalized to β-actin, and the ratio was expressed relative to the control si-GL2 sample. (C) The basal level of p21 mRNA is induced by siRNA-mediated depletion of UBE2G1 and UBE2G2 (left) or Cdt2 (right). The levels were determined as described above for panel B. (D) The half-life of p21 protein after UV irradiation is not significantly altered by knockdown of UBE2G1 and UBE2G2. Smaller quantities of protein lysates were loaded from the si-UBE2G1/G2-transfected cells to ensure that the p21 signal at 0 min was comparable to that in the si-GL2-transfected cells. Quantitation of the p21 protein and the loading control signals was performed using Scion image software. (E) The ratio of p21 to the signal from the loading control (LC) was plotted after normalizing the ratio at 0 h to 100.

Journal: Molecular and Cellular Biology

Article Title: Selective Ubiquitylation of p21 and Cdt1 by UBCH8 and UBE2G Ubiquitin-Conjugating Enzymes via the CRL4 Cdt2 Ubiquitin Ligase Complex ▿ †

doi: 10.1128/MCB.05496-11

Figure Lengend Snippet: UBE2G1 and UBE2G2 regulate p21 transcriptionally but do not affect its protein stability. (A) Basal level of p21 protein increased after knockdown of UBE2G1 or UBE2G2. The level was measured as described in the legend of Fig. 1A. (B) p21 mRNA induced by knockdown of UBE2G1 or UBE2G2. The p21 mRNA level in siRNA-transfected cells was determined by quantitative real-time PCR, normalized to β-actin, and the ratio was expressed relative to the control si-GL2 sample. (C) The basal level of p21 mRNA is induced by siRNA-mediated depletion of UBE2G1 and UBE2G2 (left) or Cdt2 (right). The levels were determined as described above for panel B. (D) The half-life of p21 protein after UV irradiation is not significantly altered by knockdown of UBE2G1 and UBE2G2. Smaller quantities of protein lysates were loaded from the si-UBE2G1/G2-transfected cells to ensure that the p21 signal at 0 min was comparable to that in the si-GL2-transfected cells. Quantitation of the p21 protein and the loading control signals was performed using Scion image software. (E) The ratio of p21 to the signal from the loading control (LC) was plotted after normalizing the ratio at 0 h to 100.

Article Snippet: Anti-p21 (C-19; Santa Cruz Biotechnology), anti-UBCH8 (Proteintech Group, Inc.), anti-UBE2G1 (Sigma), anti-β-actin (Sigma), and anti-tubulin (Santa Cruz Biotechnology) antibodies were purchased from the indicated companies.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Irradiation, Quantitation Assay, Software

UBCH8 promotes UV-induced Set8 degradation and increases the monoubiquitylation of PCNA in unstressed cells. (A) HCT116p53−/− cells transfected with siRNA against UBCH8 (top) or UBE2G1 and UBE2G2 together (bottom), irradiated with UV (20 J/m2), and collected at the indicated times by direct lysis in SDS sample buffer for Western blotting with Set8 or β-actin. Smaller quantities of protein lysates were loaded from the siUBCH8-A- or the si-UBE2G-transfected cells to ensure that the Set8 signal at 0 min was comparable to that in the si-GL2-transfected cells. (B) Immunoblot of PCNA shows ubiquitylated PCNA (Ub-PCNA) and unmodified PCNA. U2OS cells were treated with siUSP1 to detect basal PCNA monoubiquitylation along with the indicated siRNA for 72 h. (C) The histogram displays the ratio of monoubiquitylated PCNA to total PCNA normalized to the ratio in control si-GL2-transfected cells.

Journal: Molecular and Cellular Biology

Article Title: Selective Ubiquitylation of p21 and Cdt1 by UBCH8 and UBE2G Ubiquitin-Conjugating Enzymes via the CRL4 Cdt2 Ubiquitin Ligase Complex ▿ †

doi: 10.1128/MCB.05496-11

Figure Lengend Snippet: UBCH8 promotes UV-induced Set8 degradation and increases the monoubiquitylation of PCNA in unstressed cells. (A) HCT116p53−/− cells transfected with siRNA against UBCH8 (top) or UBE2G1 and UBE2G2 together (bottom), irradiated with UV (20 J/m2), and collected at the indicated times by direct lysis in SDS sample buffer for Western blotting with Set8 or β-actin. Smaller quantities of protein lysates were loaded from the siUBCH8-A- or the si-UBE2G-transfected cells to ensure that the Set8 signal at 0 min was comparable to that in the si-GL2-transfected cells. (B) Immunoblot of PCNA shows ubiquitylated PCNA (Ub-PCNA) and unmodified PCNA. U2OS cells were treated with siUSP1 to detect basal PCNA monoubiquitylation along with the indicated siRNA for 72 h. (C) The histogram displays the ratio of monoubiquitylated PCNA to total PCNA normalized to the ratio in control si-GL2-transfected cells.

Article Snippet: Anti-p21 (C-19; Santa Cruz Biotechnology), anti-UBCH8 (Proteintech Group, Inc.), anti-UBE2G1 (Sigma), anti-β-actin (Sigma), and anti-tubulin (Santa Cruz Biotechnology) antibodies were purchased from the indicated companies.

Techniques: Transfection, Irradiation, Lysis, Western Blot