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: Purified recombinant human Ube1 E1 (E-305), UbcH5a/UBE2D1 (E2-616-100), UbcH5b/UBE2D2 (E2-622-100), UbcH5c/UBE2D3 (E2-627-100), wild-type ubiquitin (U-100H), K48R ubiquitin (UM-K48R-01M), and K48-only ubiquitin (UM-K480-01M) were purchased from R&D systems.

Techniques: CRISPR, Expressing, Western Blot

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: Purified recombinant human Ube1 E1 (E-305), UbcH5a/UBE2D1 (E2-616-100), UbcH5b/UBE2D2 (E2-622-100), UbcH5c/UBE2D3 (E2-627-100), wild-type ubiquitin (U-100H), K48R ubiquitin (UM-K48R-01M), and K48-only ubiquitin (UM-K480-01M) were purchased from R&D systems.

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

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: Purified recombinant human Ube1 E1 (E-305), UbcH5a/UBE2D1 (E2-616-100), UbcH5b/UBE2D2 (E2-622-100), UbcH5c/UBE2D3 (E2-627-100), wild-type ubiquitin (U-100H), K48R ubiquitin (UM-K48R-01M), and K48-only ubiquitin (UM-K480-01M) were purchased from R&D systems.

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

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: Purified recombinant human Ube1 E1 (E-305), UbcH5a/UBE2D1 (E2-616-100), UbcH5b/UBE2D2 (E2-622-100), UbcH5c/UBE2D3 (E2-627-100), wild-type ubiquitin (U-100H), K48R ubiquitin (UM-K48R-01M), and K48-only ubiquitin (UM-K480-01M) were purchased from R&D systems.

Techniques: Transfection, Expressing, Mutagenesis, Western Blot

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: Purified recombinant human Ube1 E1 (E-305), UbcH5a/UBE2D1 (E2-616-100), UbcH5b/UBE2D2 (E2-622-100), UbcH5c/UBE2D3 (E2-627-100), wild-type ubiquitin (U-100H), K48R ubiquitin (UM-K48R-01M), and K48-only ubiquitin (UM-K480-01M) were purchased from R&D systems.

Techniques: In Vivo, Western Blot, Transfection, Mutagenesis

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: Purified recombinant human Ube1 E1 (E-305), UbcH5a/UBE2D1 (E2-616-100), UbcH5b/UBE2D2 (E2-622-100), UbcH5c/UBE2D3 (E2-627-100), wild-type ubiquitin (U-100H), K48R ubiquitin (UM-K48R-01M), and K48-only ubiquitin (UM-K480-01M) were purchased from R&D systems.

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

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: Purified recombinant human Ube1 E1 (E-305), UbcH5a/UBE2D1 (E2-616-100), UbcH5b/UBE2D2 (E2-622-100), UbcH5c/UBE2D3 (E2-627-100), wild-type ubiquitin (U-100H), K48R ubiquitin (UM-K48R-01M), and K48-only ubiquitin (UM-K480-01M) were purchased from R&D systems.

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

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: Purified recombinant human Ube1 E1 (E-305), UbcH5a/UBE2D1 (E2-616-100), UbcH5b/UBE2D2 (E2-622-100), UbcH5c/UBE2D3 (E2-627-100), wild-type ubiquitin (U-100H), K48R ubiquitin (UM-K48R-01M), and K48-only ubiquitin (UM-K480-01M) were purchased from R&D systems.

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

Journal: Cell Death & Disease

Article Title: PRPF19 facilitates colorectal cancer liver metastasis through activation of the Src-YAP1 pathway via K63-linked ubiquitination of MYL9

doi: 10.1038/s41419-023-05776-2

Figure Lengend Snippet: A , B The mRNA expression ( A ) and protein expression ( B ) of PRPF19 in normal and CRC tissues were evaluated according to the data from TCGA and CPTAC database, respectively. C The expression of PRPF19 in 12 pairs of matched adjacent normal tissues (N) and CRC tissues (T). D – F Representative images of PRPF19 IHC staining in adjacent normal tissues and CRC tissues ( D ), and the expression of PRPF19 in 125 CRC patients were analyzed based on paired Student’s t test ( E ) and Student’s t test ( F ), respectively. G – J Kaplan–Meier survival analysis of the association between PRPF19 expression and the DFS or OS rate of CRC patients in stageI-II ( G , H ) and stage III–IV ( I , J ), respectively.

Article Snippet: For in vitro ubiquitination assays, MgATP Solution (R&D Systems, B-20), UBE1 (E1) (E-305-025, R&D Systems), UbcH5c/UBE2D3 (E2) (E2-627-100, R&D Systems), PRPF19 protein (Self-laboratory purification), MYL9 protein (Proteintech, Ag7636), 10X E3 Ligase Reaction Buffer (B-71, R&D Systems), Ubiquitin Recombinant Human HA-Ubiquitin Protein (U-110-01M, R&D Systems) and ddH20 were compounded in a 25ul reaction system following the manufacturer’s instructions.

Techniques: Expressing, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: PRPF19 facilitates colorectal cancer liver metastasis through activation of the Src-YAP1 pathway via K63-linked ubiquitination of MYL9

doi: 10.1038/s41419-023-05776-2

Figure Lengend Snippet: Correlations between PRPF19 and clinicopathological features.

Article Snippet: For in vitro ubiquitination assays, MgATP Solution (R&D Systems, B-20), UBE1 (E1) (E-305-025, R&D Systems), UbcH5c/UBE2D3 (E2) (E2-627-100, R&D Systems), PRPF19 protein (Self-laboratory purification), MYL9 protein (Proteintech, Ag7636), 10X E3 Ligase Reaction Buffer (B-71, R&D Systems), Ubiquitin Recombinant Human HA-Ubiquitin Protein (U-110-01M, R&D Systems) and ddH20 were compounded in a 25ul reaction system following the manufacturer’s instructions.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: PRPF19 facilitates colorectal cancer liver metastasis through activation of the Src-YAP1 pathway via K63-linked ubiquitination of MYL9

doi: 10.1038/s41419-023-05776-2

Figure Lengend Snippet: The univariate and multivariate analyses for DFS.

Article Snippet: For in vitro ubiquitination assays, MgATP Solution (R&D Systems, B-20), UBE1 (E1) (E-305-025, R&D Systems), UbcH5c/UBE2D3 (E2) (E2-627-100, R&D Systems), PRPF19 protein (Self-laboratory purification), MYL9 protein (Proteintech, Ag7636), 10X E3 Ligase Reaction Buffer (B-71, R&D Systems), Ubiquitin Recombinant Human HA-Ubiquitin Protein (U-110-01M, R&D Systems) and ddH20 were compounded in a 25ul reaction system following the manufacturer’s instructions.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: PRPF19 facilitates colorectal cancer liver metastasis through activation of the Src-YAP1 pathway via K63-linked ubiquitination of MYL9

doi: 10.1038/s41419-023-05776-2

Figure Lengend Snippet: The univariate and multivariate analyses for OS.

Article Snippet: For in vitro ubiquitination assays, MgATP Solution (R&D Systems, B-20), UBE1 (E1) (E-305-025, R&D Systems), UbcH5c/UBE2D3 (E2) (E2-627-100, R&D Systems), PRPF19 protein (Self-laboratory purification), MYL9 protein (Proteintech, Ag7636), 10X E3 Ligase Reaction Buffer (B-71, R&D Systems), Ubiquitin Recombinant Human HA-Ubiquitin Protein (U-110-01M, R&D Systems) and ddH20 were compounded in a 25ul reaction system following the manufacturer’s instructions.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: PRPF19 facilitates colorectal cancer liver metastasis through activation of the Src-YAP1 pathway via K63-linked ubiquitination of MYL9

doi: 10.1038/s41419-023-05776-2

Figure Lengend Snippet: A The efficient overexpression and suppression of PRPF19 were confirmed by WB in the indicated stable cell lines. B , C Transwell assays showed the migration and invasion ability of HCT116 and DLD1 cells with PRPF19 overexpression or knockdown (left panel), and cell quantification was shown (right panel). D , E Representative images showed the wound healing capability of HCT116 and DLD1 cells with PRPF19 overexpression or knockdown. *** p < 0.001, ** p < 0.01, * p < 0.05 based on Student’s t test. These experiments were repeated at least three times. Error bars, mean ± SD.

Article Snippet: For in vitro ubiquitination assays, MgATP Solution (R&D Systems, B-20), UBE1 (E1) (E-305-025, R&D Systems), UbcH5c/UBE2D3 (E2) (E2-627-100, R&D Systems), PRPF19 protein (Self-laboratory purification), MYL9 protein (Proteintech, Ag7636), 10X E3 Ligase Reaction Buffer (B-71, R&D Systems), Ubiquitin Recombinant Human HA-Ubiquitin Protein (U-110-01M, R&D Systems) and ddH20 were compounded in a 25ul reaction system following the manufacturer’s instructions.

Techniques: Over Expression, Stable Transfection, Migration, Knockdown

Journal: Cell Death & Disease

Article Title: PRPF19 facilitates colorectal cancer liver metastasis through activation of the Src-YAP1 pathway via K63-linked ubiquitination of MYL9

doi: 10.1038/s41419-023-05776-2

Figure Lengend Snippet: A DLD1 cells were treated with MG132 (20 μM) for 5 h, lysed and subjected to immunoprecipitation with anti-PRPF19 antibody or IgG using silver staining. The red arrow represents the detected bands. The potential interacting proteins of PRPF19 that possessed the highest score were listed (right panel). B The DLD1 cells were co-transfected with the indicated plasmids for 48 h, and cells were treated with MG132 for 5 h. The cell lysates were immunoprecipitated with antibody anti-Flag and immunoblotted with anti-HA (left panel); immunoprecipitated with antibody anti-HA and immunoblotted with anti-Flag (right panel). C DLD1 cells were treated with MG132 for 5 h before being collected. Cell lysates were immunoprecipitated with anti-IgG, anti-MYL9, or anti-PRPF19 followed by immunoblot. D , E The mRNA ( D ) and protein ( E ) expression level of MYL9 in the DLD1 stable cell lines. F , G The indicated stable cell lines with PRPF19 knockdown (scramble/shPRPF19) or overexpression (Ctrl/PRPF19) were treated with CHX, and lysed for WB analysis at the specific time points. The intensity of MYL9 expression for each time point was quantified with GAPDH as a normalizer. *** p < 0.001, ** p < 0.01 based on Student’s t test.

Article Snippet: For in vitro ubiquitination assays, MgATP Solution (R&D Systems, B-20), UBE1 (E1) (E-305-025, R&D Systems), UbcH5c/UBE2D3 (E2) (E2-627-100, R&D Systems), PRPF19 protein (Self-laboratory purification), MYL9 protein (Proteintech, Ag7636), 10X E3 Ligase Reaction Buffer (B-71, R&D Systems), Ubiquitin Recombinant Human HA-Ubiquitin Protein (U-110-01M, R&D Systems) and ddH20 were compounded in a 25ul reaction system following the manufacturer’s instructions.

Techniques: Immunoprecipitation, Silver Staining, Transfection, Western Blot, Expressing, Stable Transfection, Knockdown, Over Expression

Journal: Cell Death & Disease

Article Title: PRPF19 facilitates colorectal cancer liver metastasis through activation of the Src-YAP1 pathway via K63-linked ubiquitination of MYL9

doi: 10.1038/s41419-023-05776-2

Figure Lengend Snippet: A , B The indicated stable cell lines with PRPF19 overexpression (Ctrl/PRPF19) or knockdown (scramble/shPRPF19) were treated with MG132 or DMSO as control. Cell lysates were analyzed by immunoblot. C HEK293T cells were transfected with siRNA of PRPF19 for 24 h, and then co-transfected with HA-MYL9 and His-Ub plasmids for another 24 h. After treatment with MG132 for 5 h, cell lysates were collected for co-IP and subjected to immunoblot analysis. D In vivo poly-ubiquitination of MYL9. HEK293T cells were co-transfected with plasmids of MYL9, PRPF19 and WT Ub or Ub mutants (K48O, K63O) for 48 h, followed by MG132 treatment for 5 h before lysis. Immunoprecipitation of ubiquitin-conjugated MYL9 protein was performed with anti-His antibody. Immuno-complexes and input were analyzed by immunoblot. E HEK293T cells were transfected with the plasmids of MYL9, PRPF19 and WT Ub or Ub mutants (K48R, K63R) for 48 h. Cells were treated with MG132 and lysed for immunoprecipitation and immunoblot as described in ( D ). F The in vitro ubiquitination assays were performed and the western blot assays with the indicated antibody. G , H Representative IHC staining images for PRPF19 and MYL9 of different staining intensities ( G ), and the correlation between the expression of PRPF19 and MYL9 based on the H-score in CRC patient tissues ( n = 30) were analyzed ( H ).

Article Snippet: For in vitro ubiquitination assays, MgATP Solution (R&D Systems, B-20), UBE1 (E1) (E-305-025, R&D Systems), UbcH5c/UBE2D3 (E2) (E2-627-100, R&D Systems), PRPF19 protein (Self-laboratory purification), MYL9 protein (Proteintech, Ag7636), 10X E3 Ligase Reaction Buffer (B-71, R&D Systems), Ubiquitin Recombinant Human HA-Ubiquitin Protein (U-110-01M, R&D Systems) and ddH20 were compounded in a 25ul reaction system following the manufacturer’s instructions.

Techniques: Stable Transfection, Over Expression, Knockdown, Control, Western Blot, Transfection, Co-Immunoprecipitation Assay, In Vivo, Ubiquitin Proteomics, Lysis, Immunoprecipitation, In Vitro, Immunohistochemistry, Staining, Expressing

Journal: Cell Death & Disease

Article Title: PRPF19 facilitates colorectal cancer liver metastasis through activation of the Src-YAP1 pathway via K63-linked ubiquitination of MYL9

doi: 10.1038/s41419-023-05776-2

Figure Lengend Snippet: A , B The mRNA expression of YAP1 and its downstream targets were detected by qRT-PCR. C , D The DLD1 cells stably overexpressed PRPF19 were transfected with siRNA of MYL9, and HCT116 cells stably interfered with PRPF19 were transfected with MYL9 plasmids pReceiver-MYL9 (pR-MYL9) or pReceiver (pR). The indicated molecules were analyzed by WB. *** p < 0.001 based on Student’s t test.

Article Snippet: For in vitro ubiquitination assays, MgATP Solution (R&D Systems, B-20), UBE1 (E1) (E-305-025, R&D Systems), UbcH5c/UBE2D3 (E2) (E2-627-100, R&D Systems), PRPF19 protein (Self-laboratory purification), MYL9 protein (Proteintech, Ag7636), 10X E3 Ligase Reaction Buffer (B-71, R&D Systems), Ubiquitin Recombinant Human HA-Ubiquitin Protein (U-110-01M, R&D Systems) and ddH20 were compounded in a 25ul reaction system following the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Stable Transfection, Transfection

Journal: Cell Death & Disease

Article Title: PRPF19 facilitates colorectal cancer liver metastasis through activation of the Src-YAP1 pathway via K63-linked ubiquitination of MYL9

doi: 10.1038/s41419-023-05776-2

Figure Lengend Snippet: PRPF19 enhanced the stability of MYL9 via K63-linked ubiquitination, and the Src-YAP1 pathway acts as the downstream effector mechanism of the PRPF19/MYL9 axis in CRC metastasis.

Article Snippet: For in vitro ubiquitination assays, MgATP Solution (R&D Systems, B-20), UBE1 (E1) (E-305-025, R&D Systems), UbcH5c/UBE2D3 (E2) (E2-627-100, R&D Systems), PRPF19 protein (Self-laboratory purification), MYL9 protein (Proteintech, Ag7636), 10X E3 Ligase Reaction Buffer (B-71, R&D Systems), Ubiquitin Recombinant Human HA-Ubiquitin Protein (U-110-01M, R&D Systems) and ddH20 were compounded in a 25ul reaction system following the manufacturer’s instructions.

Techniques: Ubiquitin Proteomics