Journal: Molecular Therapy. Nucleic Acids

Article Title: The SWIB/MDM2 motif of UBE4B activates the p53 pathway

doi: 10.1016/j.omtn.2023.02.002

Figure Lengend Snippet: Analysis of a series of UBE4B truncations (A) Schematic representation of UBE4B truncations fused to the FLAG-tagged expression vector. (B) HCT116 cells were transfected with a series of FLAG-UBE4B mutants as indicated. Cells were lysed and analyzed by immunoblotting with anti-p53 and anti-FLAG for UBE4B or various mutants of UBE4B. An antibody against β-actin was used as a loading control. (C) HCT116 cells were transfected with plasmids expressing UBE4B and UBE4BΔU as indicated. The levels of endogenous p53 were analyzed by western blotting.

Article Snippet: All PCR products were confirmed by sequencing. p53-Specific antibodies (Pab1801 and DO-1, Santa Cruz Biotechnology), Mdm2-specific antibodies (2A10, EMD Biosciences; SMP14, BD Biosciences), p21-specific antibody (F-5, Santa Cruz), Myc-specific antibody (9E10, Roche), FLAG-specific antibodies (M5 and M2, Sigma), Ub-specific antibody (P4D1, Santa Cruz), Ufd2/E4-specific antibodies (#611966, BD Biosciences), actin-specific antibody (A2228, Sigma), UBE1-specific antibody (Ab34711, Abcam), and UbcH5b-specific antibody (NBP1-81769, Novus Biologicals), and polyclonal antibodies for UBE4B (Ab97697, Abcam), anti-His (#70796-3, Novagen), anti-biotin (sc-101339), and anti-GST (B-14, Santa Cruz) were used according to the manufacturer’s instructions.

Techniques: Expressing, Plasmid Preparation, Transfection, Western Blot, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: The SWIB/MDM2 motif of UBE4B activates the p53 pathway

doi: 10.1016/j.omtn.2023.02.002

Figure Lengend Snippet: Stabilization of endogenous p53 by UBE4BΔU (A) HCT116 clones stably expressing an empty vector, UBE4B, UBE4B 1–1,000, UBE4BΔ800–1,000, and UBE4BΔU were subjected to immunoblotting with indicated antibodies. An antibody against β-actin was used as a loading control. (B) HCT116 cells were transfected with plasmids expressing UBE4B and several UBE4B mutants as indicated. Cell extracts were immunoprecipitated with an anti-FLAG-specific antibody (M5) and analyzed by immunoblotting with antibodies against p53 or FLAG (M5). (C) HCT116 cells were transiently transfected with an empty vector; 24 h later, the cells were treated with cycloheximide (CHX, 20 μg/mL) for 6 h. The levels of p53 were determined by immunoblotting with a p53-specific antibody (DO-1). An antibody against β-actin was used as a loading control. (D) Similar to (C), except that HCT116 cells were transfected with the UBE4B expression plasmid. (E) Similar to (C), except that HCT116 cells were transfected with a plasmid expressing UBE4BΔU. (F) Similar to (C), except that HCT116 cells were transfected with a plasmid expressing UBE4BΔ800–1,000. (G) Expression levels of p53 in HCT116 cells were determined by immunoblot densitometry in (C), (D), (E), and (F). Error bars indicate SEM (n = 3).

Article Snippet: All PCR products were confirmed by sequencing. p53-Specific antibodies (Pab1801 and DO-1, Santa Cruz Biotechnology), Mdm2-specific antibodies (2A10, EMD Biosciences; SMP14, BD Biosciences), p21-specific antibody (F-5, Santa Cruz), Myc-specific antibody (9E10, Roche), FLAG-specific antibodies (M5 and M2, Sigma), Ub-specific antibody (P4D1, Santa Cruz), Ufd2/E4-specific antibodies (#611966, BD Biosciences), actin-specific antibody (A2228, Sigma), UBE1-specific antibody (Ab34711, Abcam), and UbcH5b-specific antibody (NBP1-81769, Novus Biologicals), and polyclonal antibodies for UBE4B (Ab97697, Abcam), anti-His (#70796-3, Novagen), anti-biotin (sc-101339), and anti-GST (B-14, Santa Cruz) were used according to the manufacturer’s instructions.

Techniques: Clone Assay, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Transfection, Immunoprecipitation

Journal: Molecular Therapy. Nucleic Acids

Article Title: The SWIB/MDM2 motif of UBE4B activates the p53 pathway

doi: 10.1016/j.omtn.2023.02.002

Figure Lengend Snippet: The C-terminal truncations of UBE4B contribute to its ligase activity to p53 (A) HCT116, p53 −/− cells were transfected with plasmids expressing p53 or co-expression of various UBE4B constructs as indicated. Cells were lysed using RIPA buffer with SDS, immunoprecipitated with a specific antibody for p53 (Pab1801), and analyzed by immunoblotting with antibodies against p53 (upper panel, DO-1) or anti-FLAG for UBE4B constructs (lower panel, M2). β-Actin was used as a loading control. The arrow indicates the position of UBE4B mutants. (B) His-UBE4BΔ800–1,000 was evaluated for its self-ubiquitination activity in the presence of recombinant E1 (UBE1), E2 (UbcH5b), and ubiquitin (Ub) as indicated. Following the ubiquitination reaction, the samples were subjected to SDS-PAGE and immunoblotting with a His antibody to reveal ubiquitinated products. (C) His-UBE4BΔU, His-UBE4B 1,000–1,173, and His-UBE4B 1,100–1,173 fusion proteins were purified from E . coli and evaluated for their ligase activity in the presence of E1, E2, and Ub, as indicated. The self-ubiquitination of UBE4B mutants was detected by immunoblotting with an anti-His antibody to reveal ubiquitinated products. (D) His-UBE4B and various truncated His-UBE4B fusion proteins were affinity purified from E . coli and analyzed by western blotting with an anti-His antibody (top image, input). Various His-UBE4B constructs were evaluated for their ability to ubiquitinate purified p53 protein in the presence of GST-Hdm2 and analyzed by immunoblotting with a p53-specific antibody (DO-1) to detect ubiquitinated p53 (middle image). Purified E1 (UBE1), E2 (UbcH5b), and GST-Hdm2 are shown in the bottom three lanes.

Article Snippet: All PCR products were confirmed by sequencing. p53-Specific antibodies (Pab1801 and DO-1, Santa Cruz Biotechnology), Mdm2-specific antibodies (2A10, EMD Biosciences; SMP14, BD Biosciences), p21-specific antibody (F-5, Santa Cruz), Myc-specific antibody (9E10, Roche), FLAG-specific antibodies (M5 and M2, Sigma), Ub-specific antibody (P4D1, Santa Cruz), Ufd2/E4-specific antibodies (#611966, BD Biosciences), actin-specific antibody (A2228, Sigma), UBE1-specific antibody (Ab34711, Abcam), and UbcH5b-specific antibody (NBP1-81769, Novus Biologicals), and polyclonal antibodies for UBE4B (Ab97697, Abcam), anti-His (#70796-3, Novagen), anti-biotin (sc-101339), and anti-GST (B-14, Santa Cruz) were used according to the manufacturer’s instructions.

Techniques: Activity Assay, Transfection, Expressing, Construct, Immunoprecipitation, Western Blot, Control, Ubiquitin Proteomics, Recombinant, SDS Page, Purification, Affinity Purification

Journal: Molecular Therapy. Nucleic Acids

Article Title: The SWIB/MDM2 motif of UBE4B activates the p53 pathway

doi: 10.1016/j.omtn.2023.02.002

Figure Lengend Snippet: Effect of various UBE4B mutants on p53 functions (A) HCT116, p53 −/− cells were co-transfected with a reporter plasmid for p21-luciferase (Luc), and a p53 expression construct in combination with UBE4BΔ800–1,000, UBE4B, UBE4BΔU, or Hdm2 expression constructs, or an empty vector. Transcriptional activity of p53 is shown; error bars indicate SEM (n = 3). ∗∗p < 0.01; ∗∗∗∗p < 0.0001. (B) Western blot of UBE4B constructs, p53, and Hdm2 with FLAG-specific antibodies for UBE4B constructs, p53-specific (DO-1), and Myc-specific for Hdm2. (C) HCT116 stable clones expressing UBE4BΔ800–1,000, UBE4B, UBE4BΔU, or an empty vector were transiently transfected with a p21-Luc reporter plasmid, and luciferase activity was measured. Error bars indicate SEM (n = 3). ∗∗p < 0.01; ∗∗∗p < 0.001. (D) HCT116, p53 −/− cells were transfected with p53 alone or p53 in combination with UBE4B, Hdm2, various UBE4B mutants, or an empty vector (pcDNA3) as indicated, and the cell-cycle profile was determined by propidium iodide staining and flow cytometry. The results represent the average of triplicate experiments. (E) HCT116, p53 −/− cells were transfected with plasmids expressing p53 or p53 in combination with UBE4B, Hdm2, UBE4BΔ800–1,000, UBE4B 500–1,173, UBE4B 800–1,173, and UBE4B 1,000–1,100. The effect of various UBE4B constructs on p53-dependent apoptosis was determined by annexin V staining and flow cytometry. Error bars indicate SEM (n = 3). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (F) The levels of UBE4B mutants, p53, and Hdm2 expression were detected by western blotting. β-Actin was used as a loading control.

Article Snippet: All PCR products were confirmed by sequencing. p53-Specific antibodies (Pab1801 and DO-1, Santa Cruz Biotechnology), Mdm2-specific antibodies (2A10, EMD Biosciences; SMP14, BD Biosciences), p21-specific antibody (F-5, Santa Cruz), Myc-specific antibody (9E10, Roche), FLAG-specific antibodies (M5 and M2, Sigma), Ub-specific antibody (P4D1, Santa Cruz), Ufd2/E4-specific antibodies (#611966, BD Biosciences), actin-specific antibody (A2228, Sigma), UBE1-specific antibody (Ab34711, Abcam), and UbcH5b-specific antibody (NBP1-81769, Novus Biologicals), and polyclonal antibodies for UBE4B (Ab97697, Abcam), anti-His (#70796-3, Novagen), anti-biotin (sc-101339), and anti-GST (B-14, Santa Cruz) were used according to the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Luciferase, Expressing, Construct, Activity Assay, Western Blot, Clone Assay, Staining, Flow Cytometry, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: The SWIB/MDM2 motif of UBE4B activates the p53 pathway

doi: 10.1016/j.omtn.2023.02.002

Figure Lengend Snippet: Peptide-protein molecular docking (A) The location of the UBE4B peptide (top ten models with a different color) within the 3D structure of p53 DNA-binding domain (DBD) using HPEDOCK. In the upper panel we use the “cartoon” style for p53 protein and the SWIB/Hdm2 peptide; in the lower panel we use the molecular “surface” style for p53 protein and the “cartoon” style for the SWIB/Hdm2 peptide. (B) The location of a representative of the SWIB/Hdm2 peptide (model 8, purple) with the 3D structure of p53 DBD using HPEDOCK. (C) The location of a representative of the SWIB/Hdm2 peptide (model 9, green) with the 3D structure of p53 DBD using HPEDOCK. (D) The predicted binding model between the SWIB/Hdm2 motif and p53 DBD is shown in the left panel using MOE software (PDB: 2FEJ). The binding interaction of SWIB/Hdm2 with the binding site of p53 DBD is displayed in the right panel. (E) The predicted binding model between SWIB/Hdm2 motif and the p53 OD is shown in the left panel using MOE software (PDB: 1OLG). The binding interaction of SWIB/Hdm2 with the binding site of the p53 OD is displayed in the right panel. (F) The SWIB/Hdm2 motif does not bind to the transactivation domain of p53 using MOE software (PDB: 1YCR). (G) The SWIB/Hdm2 motif does not bind to mutant p53 using MOE software (PDB: 4MZI).

Article Snippet: All PCR products were confirmed by sequencing. p53-Specific antibodies (Pab1801 and DO-1, Santa Cruz Biotechnology), Mdm2-specific antibodies (2A10, EMD Biosciences; SMP14, BD Biosciences), p21-specific antibody (F-5, Santa Cruz), Myc-specific antibody (9E10, Roche), FLAG-specific antibodies (M5 and M2, Sigma), Ub-specific antibody (P4D1, Santa Cruz), Ufd2/E4-specific antibodies (#611966, BD Biosciences), actin-specific antibody (A2228, Sigma), UBE1-specific antibody (Ab34711, Abcam), and UbcH5b-specific antibody (NBP1-81769, Novus Biologicals), and polyclonal antibodies for UBE4B (Ab97697, Abcam), anti-His (#70796-3, Novagen), anti-biotin (sc-101339), and anti-GST (B-14, Santa Cruz) were used according to the manufacturer’s instructions.

Techniques: Binding Assay, Software, Mutagenesis

Journal: Molecular Therapy. Nucleic Acids

Article Title: The SWIB/MDM2 motif of UBE4B activates the p53 pathway

doi: 10.1016/j.omtn.2023.02.002

Figure Lengend Snippet: The SWIB/Hdm2 motif interacts with p53 and inhibits cell growth in WT p53 cancer cells (A) GST-p53 fusion proteins were purified and analyzed by immunoblotting with a GST-specific antibody. (B) A biotinylated peptide pull-down assay was performed. Five microliters of biotinylated peptide (1.25 μg) was mixed with 2–4 μg of GST-p53 fusion proteins, and incubated at 4°C for 4 h. Streptavidin Sepharose beads were then added and incubated at 4°C for 1 h, washed, and analyzed by immunoblotting with an antibody against GST. (C) Similar to (B), except that the biotinylated control peptide was added. (D) His-UBE4B was evaluated for its E3 activity in the presence of recombinant E1, E2 (UbcH5b), Ub, p53, and the SWIB/Hdm2 peptide. Following the ubiquitination reaction, the samples were subjected to SDS-PAGE and immunoblotting with antibodies against p53 (DO-1) to reveal ubiquitinated p53, or E1 (UBE1) and E2 (UbcH5b) as indicated. (E) The SWIB/Hdm2 peptide does not block to form thioester conjugates with HA-Ub-E1 or HA-Ub-UbcH5b. The samples were subjected to nonreducing SDS-PAGE and analyzed by western blotting with an HA-specific antibody. (F) HCT116 cells were treated with 20 μM SWIB/Hdm2 peptide or control peptide for 40 h in the presence of 20 μM MG132, a proteasome inhibitor, as indicated, immunoprecipitated with a p53-specific antibody (Pab1801) or a control antibody (immunoglobulin G), and analyzed by western blotting with antibodies to detect UBE4B or p53. Western blots for p53, UBE4B, and actin are shown in the lower panels. (G) Similar to (F), except that an anti-UBE4B antibody was used. (H) HCT116 cells were treated with the control peptide or the SWIB/Hdm2 peptide at indicated concentrations for 40 h. Cell viability was examined by the MTT assay. (I) Western blot of HCT116 cells treated with different concentrations of the control peptide probed with antibodies against p53 (DO-1), UBE4B, and actin, as indicated. (J) Similar to (I), except that HCT116 cells were treated with the SWIB/Hdm2 peptide. (K) Similar to (H), except that HCT116, p53 −/− cells were used. (L) Similar to (I), except that HCT116, p53 −/− cells were used. (M) Similar to (J), except that HCT116, p53 −/− cells were used.

Article Snippet: All PCR products were confirmed by sequencing. p53-Specific antibodies (Pab1801 and DO-1, Santa Cruz Biotechnology), Mdm2-specific antibodies (2A10, EMD Biosciences; SMP14, BD Biosciences), p21-specific antibody (F-5, Santa Cruz), Myc-specific antibody (9E10, Roche), FLAG-specific antibodies (M5 and M2, Sigma), Ub-specific antibody (P4D1, Santa Cruz), Ufd2/E4-specific antibodies (#611966, BD Biosciences), actin-specific antibody (A2228, Sigma), UBE1-specific antibody (Ab34711, Abcam), and UbcH5b-specific antibody (NBP1-81769, Novus Biologicals), and polyclonal antibodies for UBE4B (Ab97697, Abcam), anti-His (#70796-3, Novagen), anti-biotin (sc-101339), and anti-GST (B-14, Santa Cruz) were used according to the manufacturer’s instructions.

Techniques: Purification, Western Blot, Pull Down Assay, Incubation, Control, Activity Assay, Recombinant, Ubiquitin Proteomics, SDS Page, Blocking Assay, Immunoprecipitation, MTT Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: The SWIB/MDM2 motif of UBE4B activates the p53 pathway

doi: 10.1016/j.omtn.2023.02.002

Figure Lengend Snippet: The SWIB/Hdm2 peptide activates p53 (A) HCT116 cells were treated with the control peptide or the SWIB/Hdm2 peptide (0 and 20 μM). The effect of the SWIB/Hdm2 peptide on p53-dependent apoptosis was determined by annexin V staining and flow cytometry. Error bars indicate SEM (n = 3). ∗∗p < 0.01. (B) HCT116 cells were treated with the control peptide or the SWIB/Hdm2 peptide as indicated. Propidium iodide staining and flow cytometry (left panel) were used to determine the cell-cycle profile. The results represent the average of three experiments in triplicate. The G 1 /S ratio is provided. (C) HCT116, p53 −/− cells were transfected with plasmids expressing an empty vector, or p53, or combined with UBE4B or UBE4BΔU. At 30 h post transfection, cells were split, treated with the control peptide or the SWIB/Hdm2 peptide, and selected with G418, and colonies were stained after 2 weeks. The numbers were counted and plotted. All experiments were performed in triplicate. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (D) Representative colonies are shown as indicated. All experiments were performed in triplicate. (E) Similar to (A), except that HCT116, p53 −/− cells were transfected with the indicated expression plasmids. Annexin V staining and flow cytometry were performed to detect cell apoptosis. ∗p < 0.05; ∗∗∗p < 0.001. (F) Similar to (B), except that HCT116, p53 −/− cells were transfected with the indicated expression plasmids. (G) Protein expression levels of UBE4B, UBE4BΔU, p53, and p21 were measured by western blotting. β-Actin was used as a loading control.

Article Snippet: All PCR products were confirmed by sequencing. p53-Specific antibodies (Pab1801 and DO-1, Santa Cruz Biotechnology), Mdm2-specific antibodies (2A10, EMD Biosciences; SMP14, BD Biosciences), p21-specific antibody (F-5, Santa Cruz), Myc-specific antibody (9E10, Roche), FLAG-specific antibodies (M5 and M2, Sigma), Ub-specific antibody (P4D1, Santa Cruz), Ufd2/E4-specific antibodies (#611966, BD Biosciences), actin-specific antibody (A2228, Sigma), UBE1-specific antibody (Ab34711, Abcam), and UbcH5b-specific antibody (NBP1-81769, Novus Biologicals), and polyclonal antibodies for UBE4B (Ab97697, Abcam), anti-His (#70796-3, Novagen), anti-biotin (sc-101339), and anti-GST (B-14, Santa Cruz) were used according to the manufacturer’s instructions.

Techniques: Control, Staining, Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: The SWIB/MDM2 motif of UBE4B activates the p53 pathway

doi: 10.1016/j.omtn.2023.02.002

Figure Lengend Snippet: Model of p53 regulation and targeting the p53-UBE4B interaction p53 is tightly regulated by Ub E3 ligases and is maintained at a low level under unstressed conditions. UBE4B, Hdm2, and p53 form a complex that promotes polyubiquitination and degradation of p53. The SWIB/Hdm2 motif derived from UBE4B was shown to bind to p53, reduce UBE4B-mediated ubiquitination of p53, and activate p53.

Article Snippet: All PCR products were confirmed by sequencing. p53-Specific antibodies (Pab1801 and DO-1, Santa Cruz Biotechnology), Mdm2-specific antibodies (2A10, EMD Biosciences; SMP14, BD Biosciences), p21-specific antibody (F-5, Santa Cruz), Myc-specific antibody (9E10, Roche), FLAG-specific antibodies (M5 and M2, Sigma), Ub-specific antibody (P4D1, Santa Cruz), Ufd2/E4-specific antibodies (#611966, BD Biosciences), actin-specific antibody (A2228, Sigma), UBE1-specific antibody (Ab34711, Abcam), and UbcH5b-specific antibody (NBP1-81769, Novus Biologicals), and polyclonal antibodies for UBE4B (Ab97697, Abcam), anti-His (#70796-3, Novagen), anti-biotin (sc-101339), and anti-GST (B-14, Santa Cruz) were used according to the manufacturer’s instructions.

Techniques: Derivative Assay, Ubiquitin Proteomics

Journal: Molecular Therapy. Nucleic Acids

Article Title: The SWIB/MDM2 motif of UBE4B activates the p53 pathway

doi: 10.1016/j.omtn.2023.02.002

Figure Lengend Snippet: Effect of various UBE4B mutants on p53 functions (A) HCT116, p53 −/− cells were co-transfected with a reporter plasmid for p21-luciferase (Luc), and a p53 expression construct in combination with UBE4BΔ800–1,000, UBE4B, UBE4BΔU, or Hdm2 expression constructs, or an empty vector. Transcriptional activity of p53 is shown; error bars indicate SEM (n = 3). ∗∗p < 0.01; ∗∗∗∗p < 0.0001. (B) Western blot of UBE4B constructs, p53, and Hdm2 with FLAG-specific antibodies for UBE4B constructs, p53-specific (DO-1), and Myc-specific for Hdm2. (C) HCT116 stable clones expressing UBE4BΔ800–1,000, UBE4B, UBE4BΔU, or an empty vector were transiently transfected with a p21-Luc reporter plasmid, and luciferase activity was measured. Error bars indicate SEM (n = 3). ∗∗p < 0.01; ∗∗∗p < 0.001. (D) HCT116, p53 −/− cells were transfected with p53 alone or p53 in combination with UBE4B, Hdm2, various UBE4B mutants, or an empty vector (pcDNA3) as indicated, and the cell-cycle profile was determined by propidium iodide staining and flow cytometry. The results represent the average of triplicate experiments. (E) HCT116, p53 −/− cells were transfected with plasmids expressing p53 or p53 in combination with UBE4B, Hdm2, UBE4BΔ800–1,000, UBE4B 500–1,173, UBE4B 800–1,173, and UBE4B 1,000–1,100. The effect of various UBE4B constructs on p53-dependent apoptosis was determined by annexin V staining and flow cytometry. Error bars indicate SEM (n = 3). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (F) The levels of UBE4B mutants, p53, and Hdm2 expression were detected by western blotting. β-Actin was used as a loading control.

Article Snippet: All PCR products were confirmed by sequencing. p53-Specific antibodies (Pab1801 and DO-1, Santa Cruz Biotechnology), Mdm2-specific antibodies (2A10, EMD Biosciences; SMP14, BD Biosciences), p21-specific antibody (F-5, Santa Cruz), Myc-specific antibody (9E10, Roche), FLAG-specific antibodies (M5 and M2, Sigma), Ub-specific antibody (P4D1, Santa Cruz), Ufd2/E4-specific antibodies (#611966, BD Biosciences), actin-specific antibody (A2228, Sigma), UBE1-specific antibody (Ab34711, Abcam), and UbcH5b-specific antibody (NBP1-81769, Novus Biologicals), and polyclonal antibodies for UBE4B (Ab97697, Abcam), anti-His (#70796-3, Novagen), anti-biotin (sc-101339), and anti-GST (B-14, Santa Cruz) were used according to the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Luciferase, Expressing, Construct, Activity Assay, Western Blot, Clone Assay, Staining, Flow Cytometry, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: The SWIB/MDM2 motif of UBE4B activates the p53 pathway

doi: 10.1016/j.omtn.2023.02.002

Figure Lengend Snippet: The SWIB/Hdm2 peptide activates p53 (A) HCT116 cells were treated with the control peptide or the SWIB/Hdm2 peptide (0 and 20 μM). The effect of the SWIB/Hdm2 peptide on p53-dependent apoptosis was determined by annexin V staining and flow cytometry. Error bars indicate SEM (n = 3). ∗∗p < 0.01. (B) HCT116 cells were treated with the control peptide or the SWIB/Hdm2 peptide as indicated. Propidium iodide staining and flow cytometry (left panel) were used to determine the cell-cycle profile. The results represent the average of three experiments in triplicate. The G 1 /S ratio is provided. (C) HCT116, p53 −/− cells were transfected with plasmids expressing an empty vector, or p53, or combined with UBE4B or UBE4BΔU. At 30 h post transfection, cells were split, treated with the control peptide or the SWIB/Hdm2 peptide, and selected with G418, and colonies were stained after 2 weeks. The numbers were counted and plotted. All experiments were performed in triplicate. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (D) Representative colonies are shown as indicated. All experiments were performed in triplicate. (E) Similar to (A), except that HCT116, p53 −/− cells were transfected with the indicated expression plasmids. Annexin V staining and flow cytometry were performed to detect cell apoptosis. ∗p < 0.05; ∗∗∗p < 0.001. (F) Similar to (B), except that HCT116, p53 −/− cells were transfected with the indicated expression plasmids. (G) Protein expression levels of UBE4B, UBE4BΔU, p53, and p21 were measured by western blotting. β-Actin was used as a loading control.

Article Snippet: All PCR products were confirmed by sequencing. p53-Specific antibodies (Pab1801 and DO-1, Santa Cruz Biotechnology), Mdm2-specific antibodies (2A10, EMD Biosciences; SMP14, BD Biosciences), p21-specific antibody (F-5, Santa Cruz), Myc-specific antibody (9E10, Roche), FLAG-specific antibodies (M5 and M2, Sigma), Ub-specific antibody (P4D1, Santa Cruz), Ufd2/E4-specific antibodies (#611966, BD Biosciences), actin-specific antibody (A2228, Sigma), UBE1-specific antibody (Ab34711, Abcam), and UbcH5b-specific antibody (NBP1-81769, Novus Biologicals), and polyclonal antibodies for UBE4B (Ab97697, Abcam), anti-His (#70796-3, Novagen), anti-biotin (sc-101339), and anti-GST (B-14, Santa Cruz) were used according to the manufacturer’s instructions.

Techniques: Control, Staining, Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Western Blot