Journal: The Journal of Biological Chemistry

Article Title: Human epidermal growth factor receptor 4 (Her4) Suppresses p53 Protein via Targeting the MDMX-MDM2 Protein Complex

doi: 10.1074/jbc.M116.752303

Figure Lengend Snippet: Her4 expression increases the interaction between MDMX and MDM2 and suppresses p53 activity. A, HEK293T cells were transfected with a combination of expression plasmids for Her4, MDMX, and/or MDM2 with a control GFP vector. The cell lysates were then blotted for the indicated antibodies. L.E., low exposure; H.E., high exposure. B, HEK293T cells were transfected with a combination of expression vectors for MDMX, FLAG-MDM2, and/or Her4 and then harvested 24 h later for IP with FLAG antibody-conjugated beads. Whole cell extracts (WCE) and FLAG IP samples were subjected to Western blotting analysis with the indicated antibodies. C, MCF-7 cells were treated with or without NRG1 for 24 h (DMEM/1% FBS), and then cell lysates were used for MDM2 immunoprecipitation using MDM2-agarose-conjugated beads. Whole cell extracts and IP were subjected to Western blotting analysis with the indicated antibodies. D, U2OS cells were transfected with the Her4 expression vector and a p53 responsive element-driven LacZ plasmid to determine endogenous p53 transcriptional activity in response to 4-Gy irradiation (n = 3). E, MCF-7 cells were transfected with a plasmid or with various amounts of Her4 expression vector along with a pg13 luciferase reporter vector, containing 13 copies of the p53-binding consensus sequence, to determine endogenous p53 transcriptional activity under normal conditions (n = 2). Data are shown as the mean ± S.D. *, p < 0.05; t test.

Article Snippet: Membranes were probed with the following antibodies as indicated: anti-MDMX (BL1258, Bethyl Laboratories), anti-MDM2 (Santa Cruz Biotechnology), anti-Her4 (Cell Signaling Technology), anti-phospho-Her4 (Millipore), anti-FLAG M2 (Sigma), anti-p53 (Cell Signaling Technology), anti-phospho-tyrosine (9411, Cell Signaling Technology), and anti-β-actin (AC-15, Sigma).

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Irradiation, Luciferase, Binding Assay, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Human epidermal growth factor receptor 4 (Her4) Suppresses p53 Protein via Targeting the MDMX-MDM2 Protein Complex

doi: 10.1074/jbc.M116.752303

Figure Lengend Snippet: Cellular localization of wild-type MDMX, mutant MDMX S314A, MDM2, and p53. A, U2OS cells were transfected with various combinations of MDMX, MDM2, and/or Her4 and then immunostained for MDMX (GFP) and MDM2 (RFP), and the nuclei were stained with DAPI. B, U2OS cells were transfected with various combinations of GFP-p53(Δ92–112), MDMX, MDM2, and/or Her4 and then immunostained for MDM2 (RFP), and the nuclei were stained with DAPI.

Article Snippet: Membranes were probed with the following antibodies as indicated: anti-MDMX (BL1258, Bethyl Laboratories), anti-MDM2 (Santa Cruz Biotechnology), anti-Her4 (Cell Signaling Technology), anti-phospho-Her4 (Millipore), anti-FLAG M2 (Sigma), anti-p53 (Cell Signaling Technology), anti-phospho-tyrosine (9411, Cell Signaling Technology), and anti-β-actin (AC-15, Sigma).

Techniques: Mutagenesis, Transfection, Staining

Journal: The Journal of Biological Chemistry

Article Title: Human epidermal growth factor receptor 4 (Her4) Suppresses p53 Protein via Targeting the MDMX-MDM2 Protein Complex

doi: 10.1074/jbc.M116.752303

Figure Lengend Snippet: Her4 activation after NRG1 treatment increases MDMX levels, leading to the stabilization of MDM2. A, MCF-7 cells were treated with 50 ng/ml of NRG1 for Her4 activation or EGF for EGFR activation over a course of 3 h and then harvested for Western blotting analysis with the indicated antibodies. B, untreated MCF-7 and MCF-10A cells were compared for total Her4 and tubulin by Western blotting analysis (top panel). MCF-10A cells (low endogenous expression of Her4) were treated with 50 ng/ml NRG1 for 6, 12, and 24 h, followed by Western blotting analysis of the cell lysates with the indicated antibodies (bottom panel). C, MCF-7 cells were pretreated with or without γ-secretase inhibitor for 45 min, followed by 6 h of NRG1 treatment. Western blotting analysis was carried out with the indicated antibodies. D, early time points. MCF-7 cells expressing endogenous Her4 were treated with 50 ng/ml of NRG1 for 20 and 40 min, and then extracted lysates were subjected to Western blotting analysis with the indicated antibodies. E, MCF-7 cells were treated with 50 ng/ml NRG1 for 30 min, 1 h, and 12 h. The lysates were then subjected to Western blotting analysis with the indicated antibodies. F, late time points. MCF-7 cells were treated with 50 ng/ml of NRG1 for 6, 12, 36, and 48 h, followed by Western blotting analysis with the indicated antibodies. G, MCF-7 cells were treated with NRG1 for 6, 12, or 24 h. RNA samples were collected for RT-qPCR analysis of MDMX expression. Data are shown as the mean ± S.D. NS, not significant. H, MCF-7 cells were treated with NRG1 or solvent (control condition) for 24 h in DMEM/10% FBS, washed, and cultured with fresh medium containing cycloheximide (CHX, 10 μg/ml) to inhibit protein synthesis. The cells were harvested at the indicated time for Western blotting analysis to monitor the disappearance of MDMX. A–G, cells were treated with or without NRG1/EGF in DMEM/1% FBS.

Article Snippet: Membranes were probed with the following antibodies as indicated: anti-MDMX (BL1258, Bethyl Laboratories), anti-MDM2 (Santa Cruz Biotechnology), anti-Her4 (Cell Signaling Technology), anti-phospho-Her4 (Millipore), anti-FLAG M2 (Sigma), anti-p53 (Cell Signaling Technology), anti-phospho-tyrosine (9411, Cell Signaling Technology), and anti-β-actin (AC-15, Sigma).

Techniques: Activation Assay, Western Blot, Expressing, Quantitative RT-PCR, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Human epidermal growth factor receptor 4 (Her4) Suppresses p53 Protein via Targeting the MDMX-MDM2 Protein Complex

doi: 10.1074/jbc.M116.752303

Figure Lengend Snippet: Her4 decreases the susceptibility of cells to DNA damage-induced p53 pathways. A, MCF-7 cells were transfected with an empty vector or a Her4 constitutively active vector for 48 h and then irradiated with a 3-Gy dose. Protein samples were harvested 3 h post-irradiation. L.E., low exposure; H.E., high exposure. B, MCF-7 cells were pretreated with or without 50 ng/ml NRG1 for 48 h and then harvested 3 h after treatment with the indicated levels of radiation. C, MCF-7 cells were pretreated with or without 50 ng/ml NRG1 for 9 h and then treated for 3, 6, or 9 h with 375 μm 5′FU. D, MCF-7 cells were pretreated with 50 ng/ml of NRG1 or EGF for 9 h and then treated with 9 h of 375 μm 5′FU. Western blotting analysis was carried out with the indicated antibodies. E, MCF-7 cells were pretreated with or without 50 ng/ml NRG1 for 48 h. The RNA samples were collected 3 h after a 3-Gy radiation dose, followed by RT-qPCR analysis of MDM2 and p21 mRNA expression. Data are shown as the mean ± S.D. *, p < 0.05; t test; n = 3. Cells were treated with or without NRG1/EGF in DMEM/1% FBS. NT, not treated; Ctl, control.

Article Snippet: Membranes were probed with the following antibodies as indicated: anti-MDMX (BL1258, Bethyl Laboratories), anti-MDM2 (Santa Cruz Biotechnology), anti-Her4 (Cell Signaling Technology), anti-phospho-Her4 (Millipore), anti-FLAG M2 (Sigma), anti-p53 (Cell Signaling Technology), anti-phospho-tyrosine (9411, Cell Signaling Technology), and anti-β-actin (AC-15, Sigma).

Techniques: Transfection, Plasmid Preparation, Irradiation, Western Blot, Quantitative RT-PCR, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Human epidermal growth factor receptor 4 (Her4) Suppresses p53 Protein via Targeting the MDMX-MDM2 Protein Complex

doi: 10.1074/jbc.M116.752303

Figure Lengend Snippet: Her4 stabilizes MDMX through the phosphorylation of Ser-314, leading to MDM2 stabilization and p53 degradation. A, i, peptide count of the phosphosites Ser-314 and Ser-367 of MDMX in FLAG-MDMX and MDM2 versus FLAG-MDMX-, MDM2-, and Her4-transfected HEK293T cells (left panel, # designates the phosphorylation site to the left amino acid). Right panel, representative MS-MS spectrum sample of the phosphorylation event on MDMX Ser-314. ii, MDMX phosphorylation map localizing various sites, including the reported site Ser-367 (red) and the novel phosphosite Ser-314 (yellow). B, HEK293T cells were transfected with various combinations of expression vectors for wild-type MDMX, mutant MDMX S314A, MDM2, and Her4 and then harvested 48 h later for Western blotting analysis using the indicated antibodies. L.E., low exposure; H.E., high exposure. C, HEK293T cells were transfected for 24 h with either wild-type MDMX or mutant MDMX S314A with or without overexpression of Her4. Cell lysates were incubated with GST-MDM2 and then pulled down by GST beads. To compare binding affinity, the amount of MDMX protein was adjusted so comparable abundances of MDMX protein in Her4-transfected and non-transfected cells were used to incubate with GST-MDM2. Western blotting analysis was carried out with the indicated antibodies. D, MCF-7 cells were transfected with or without 0.5 or 1 μg Her4 and then harvested after 24 h for Western blotting analysis with the indicated antibodies. E, U2OS cells were transfected with various combinations of expression vectors for wild-type MDMX, mutant MDMX S314A, and Her4 and then harvested after 24 h for Western blotting analysis using the indicated antibodies.

Article Snippet: Membranes were probed with the following antibodies as indicated: anti-MDMX (BL1258, Bethyl Laboratories), anti-MDM2 (Santa Cruz Biotechnology), anti-Her4 (Cell Signaling Technology), anti-phospho-Her4 (Millipore), anti-FLAG M2 (Sigma), anti-p53 (Cell Signaling Technology), anti-phospho-tyrosine (9411, Cell Signaling Technology), and anti-β-actin (AC-15, Sigma).

Techniques: Transfection, Tandem Mass Spectroscopy, Expressing, Mutagenesis, Western Blot, Over Expression, Incubation, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Human epidermal growth factor receptor 4 (Her4) Suppresses p53 Protein via Targeting the MDMX-MDM2 Protein Complex

doi: 10.1074/jbc.M116.752303

Figure Lengend Snippet: Inhibition of CDK4/6 kinase activity prevents an Her4-mediated increase in MDMX-MDM2 and resistance to DNA-damaging activation of p53. A, prediction analysis identified only two kinases with high stringency for phosphorylating the MDMX Ser-314 site, which includes the GPS score (# designates the phosphorylation site to the left of serine. B, MCF-7 cells were pretreated either with or without CDK4/6 inhibitor IV (CDKi) for 24 h and then treated with 50 ng/ml NRG1 for 6 h. C, MCF-7 cells were pretreated either with or without doramapimod, a p38 inhibitor (p38i), for 24 h and then treated with 50 ng/ml NRG1 for 6 h. D, a kinase assay was carried out using immunopurified FLAG-tagged wild-type MDMX or mutant MDMX S314A incubated with cell lysates expressing constitutively active CDK4 or kinase-dead (K.D.) CDK4 in the presence of ATP. The products were analyzed by Western blotting analysis using anti-phosphoserine antibody (top panel) and anti-MDMX antibody (bottom panel). E, MCF-7 cells were treated for 24 h with or without 5 μm CDK4/6 inhibitor IV, followed by 6-h treatment with 50 ng/ml NRG1. Cells were then irradiated with 3 Gy, and RNA samples were collected 3 h after irradiation for RT-qPCR analysis of MDM2 and p21 mRNA expression. Data are shown as the mean ± S.D. *, p < 0.05; t test comparing treatments versus control; n = 3. Cells were treated with or without NRG1 in DMEM/1% FBS.

Article Snippet: Membranes were probed with the following antibodies as indicated: anti-MDMX (BL1258, Bethyl Laboratories), anti-MDM2 (Santa Cruz Biotechnology), anti-Her4 (Cell Signaling Technology), anti-phospho-Her4 (Millipore), anti-FLAG M2 (Sigma), anti-p53 (Cell Signaling Technology), anti-phospho-tyrosine (9411, Cell Signaling Technology), and anti-β-actin (AC-15, Sigma).

Techniques: Inhibition, Activity Assay, Activation Assay, Kinase Assay, Mutagenesis, Incubation, Expressing, Western Blot, Irradiation, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Human epidermal growth factor receptor 4 (Her4) Suppresses p53 Protein via Targeting the MDMX-MDM2 Protein Complex

doi: 10.1074/jbc.M116.752303

Figure Lengend Snippet: A working model for Her4-mediated MDMX-MDM2 complex stabilization and suppression of p53 activity. Stimulated Her4 is cleaved and activated to induce CDK4/6-dependent phosphorylation of MDMX on the Ser-314 site, leading to increased levels of MDMX, which binds to and stabilizes MDM2 and, thus, the MDMX-MDM2 complex. When stabilized, the MDMX-MDM2 heterocomplex binds to p53, suppressing its transcriptional activity and/or enhancing its ubiquitination and subsequent proteasomal degradation in the cytoplasm.

Article Snippet: Membranes were probed with the following antibodies as indicated: anti-MDMX (BL1258, Bethyl Laboratories), anti-MDM2 (Santa Cruz Biotechnology), anti-Her4 (Cell Signaling Technology), anti-phospho-Her4 (Millipore), anti-FLAG M2 (Sigma), anti-p53 (Cell Signaling Technology), anti-phospho-tyrosine (9411, Cell Signaling Technology), and anti-β-actin (AC-15, Sigma).

Techniques: Activity Assay

Journal: Molecular medicine reports

Article Title: The role of neuregulin4 and HER4 in gastrointestinal malignant lymphoma.

doi: 10.3892/mmr.2011.542

Figure Lengend Snippet: Figure 1. Analysis of EGF and HER family mRNA expression in gastric cancer, colon cancer and malignant lymphoma cell lines. A higher level of expression of HER4 and NRG4 mRNA was detected in lymphoma cell lines compared to gastric and colon cell lines. The expression of EGF and HER family genes in 4 lymphoma, 1 gastric cancer and 1 colon cancer cell line was analyzed using RT-PCR with specific oligonucleotide primer sets followed by agarose gel electrophoresis.

Article Snippet: We purchased the following antibodies: Rabbit anti-NRG4 polyclonal antibody (ab60090; Abcam, Cambridge, MA, USA), rabbit anti-HER4 polyclonal antibody (RB-9045-R7; Thermo Fisher Scientific, Worcester, MA, USA), mouse anti-HER4 monoclonal antibody (sc-8050; Santa Cruz Biotechnology, Delaware Avenue, CA, USA) and mouse anti-phospho-tyrosine, clone 4G10 (05-321; Millipore, Billerica, MA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

Journal: Molecular medicine reports

Article Title: The role of neuregulin4 and HER4 in gastrointestinal malignant lymphoma.

doi: 10.3892/mmr.2011.542

Figure Lengend Snippet: Figure 2. Immunohistochemical staining of DLBCL and FL with an anti body (A and C) against NRG4 or (B and D) against HER4.

Article Snippet: We purchased the following antibodies: Rabbit anti-NRG4 polyclonal antibody (ab60090; Abcam, Cambridge, MA, USA), rabbit anti-HER4 polyclonal antibody (RB-9045-R7; Thermo Fisher Scientific, Worcester, MA, USA), mouse anti-HER4 monoclonal antibody (sc-8050; Santa Cruz Biotechnology, Delaware Avenue, CA, USA) and mouse anti-phospho-tyrosine, clone 4G10 (05-321; Millipore, Billerica, MA, USA).

Techniques: Immunohistochemical staining, Staining

Journal: Molecular medicine reports

Article Title: The role of neuregulin4 and HER4 in gastrointestinal malignant lymphoma.

doi: 10.3892/mmr.2011.542

Figure Lengend Snippet: Figure 3. Analysis of NRG4 induction of HER4 tyrosine phosphorylation. Two kinds of lymphoma cell lines were treated with 1 µg/ml of recombinant NRG4 for 30 min. HER4 was immunoprecipitated from extracts of the lym phoma cells with an anti-HER4 antibody and was subsequently divided into two equal aliquots. One aliquot was subjected to Western blotting with an anti-P-Tyr antibody, and the second aliquot was subjected to Western blotting with an anti-HER4 loading control antibody.

Article Snippet: We purchased the following antibodies: Rabbit anti-NRG4 polyclonal antibody (ab60090; Abcam, Cambridge, MA, USA), rabbit anti-HER4 polyclonal antibody (RB-9045-R7; Thermo Fisher Scientific, Worcester, MA, USA), mouse anti-HER4 monoclonal antibody (sc-8050; Santa Cruz Biotechnology, Delaware Avenue, CA, USA) and mouse anti-phospho-tyrosine, clone 4G10 (05-321; Millipore, Billerica, MA, USA).

Techniques: Phospho-proteomics, Recombinant, Immunoprecipitation, Western Blot, Control