Journal: Cell Death & Disease

Article Title: Increased expression of EHF via gene amplification contributes to the activation of HER family signaling and associates with poor survival in gastric cancer

doi: 10.1038/cddis.2016.346

Figure Lengend Snippet: EHF is identified as a new HER2 transcription factor and the modulator of HER3 and HER4 in gastric cancer. ( a ) BGC823 cells were transiently transfected with pGL3-Basic or luciferase reporter constructs containing various lengths of the promoter region of HER2 gene, as indicated (F1: −607/+11; F2: −175/+11; F3: −607/−175) (left panels). Cotransfection with empty vector was used as a control. The ratio of the Luc/Renilla activity is shown as means±S.E. of three independent assays (right panels). ( b ) The luciferase reporter gene assay was performed to evaluate the effect of EHF knockdown on promoter activity of HER2 in BGC823 cells. The ratio of the Luc/Renilla activity is shown as means±S.E. of three independent assays. ( c ) HEK293T cells were cotransfected pGL3-HER2-Luc-F1 and various amounts of pcDNA3.1(-)A-EHF or empty vector, respectively. Promoter activities of HER2 were measured by luciferase reporter gene assays. All the ratio of the Luc/Renilla activity is shown as means±S.E. of three independent assays. ( d ) Putative promoter regions of HER2 (−607/+11), HER3 (−997/+440) and HER4 (−697/+306) were inserted into the pGL3-Basic to construct the luciferase reporter plasmid pGL3-HER2-Luc, pGL3-HER3-Luc and pGL3-HER4-Luc (upper panels). P1-P7 represent the regions analyzed by ChIP assays for HER2 , HER3 and HER4 , respectively. BGC823 cells were transiently transfected with pcDNA3.1/myc-His(-)A-EHF or empty vector, and were subjected to ChIP-qRT-PCR assays using anti-Myc tag antibody. Flod enrichment was shown as means±S.E. of three independent assays (lower panels). ( e ) EMSA assay was performed to confirm the interaction between EHF and HER2 promoter. Shown are specific DNA-binding of in vitro translated EHF protein to an oligonucleotide sequence (SH2) containing ETS responsive element (GAGGAA) from the HER2 promoter. Unlabeled mutated probes contain specific mutations in the GGAA ETS core or flanking nucleotides of core sequence, as indicated by MT1 and MT2. Unlabeled wild-type (WT) and mutated (MT1 or MT2) competitor probes were added at 100-fold molar excess. Statistically significant differences were indicated: * P <0.05; ** P <0.01; *** P <0.001

Article Snippet: The membranes were blocked for 2 h in 5% bovine serum albumin (BSA) in 1 × TBS-T (0.5% Tween-20) and incubated with the indicated primary antibodies, including anti-EHF (Abcam, Inc), anti-total-Erk1/2 (Abcam, Inc), anti-phospho-Erk1/2 (Epitomics, Inc), anti-phospho-AktSer473 (Bioworld Technology, co, Ltd), anti-total-Akt (Bioworld Technology, co, Ltd), anti-HER2 (Sino Biological, Inc), anti-HER3 (Sino Biological, Inc), anti-HER4 (Sino Biological, Inc), anti-E-cadherin (Epitomics, Inc), anti-Vimentin (Epitomics, Inc) and anti-GAPDH (Abgent, Inc).

Techniques: Transfection, Luciferase, Construct, Cotransfection, Plasmid Preparation, Activity Assay, Reporter Gene Assay, Quantitative RT-PCR, Binding Assay, In Vitro, Sequencing

Journal: Frontiers in Neuroscience

Article Title: Neuregulin1-ErbB4 Signaling in Spinal Cord Participates in Electroacupuncture Analgesia in Inflammatory Pain

doi: 10.3389/fnins.2021.636348

Figure Lengend Snippet: NRG1 effectively reduces pain hypersensitivity in CFA or Carrageenan-treated mice. (A) Experimental design of NRG1, ecto-ErbB4 or AG1478 intrathecally injected into spinal cord after CFA injection. Pain-related behaviors were tested from day –1 to day 7. (B,C) The mechanical withdrawal threshold (B) and the paw withdrawal latency (C) in mice treated with Saline, CFA + vehicle, CFA + NRG1 and CFA + ecto-ErbB4 + NRG1 group. * p < 0.05, ** p < 0.01 vs. the CFA + vehicle group. # p < 0.05, ## p < 0.01 vs. the CFA + NRG1 group. (D,E) The mechanical withdrawal threshold (D) and the paw withdrawal latency (E) in mice treated with Saline, Carrageenan + vehicle, Carrageenan + NRG1 and Carrageenan + AG1478 + NRG1 group. * p < 0.05, ** p < 0.01 vs. the Carrageenan + vehicle group; # p < 0.05, ## p < 0.01 vs. the Carrageenan + NRG1 group. (B–E) n = 10 mice per group. Data are presented as means ± SEM.

Article Snippet: The membranes were blocked with 5% non-fat milk for 1 h at RT before being probed with the primary antibodies: rabbit anti-ErbB4 (1:1000, Cell Signaling Technology, United States), mouse anti- ErbB4 (1: 500, Santa Cruz Biotechnology, United States), or rabbit anti-Neuregulin-1 (1: 500, Santa Cruz Biotechnology, United States) at 4°C overnight followed by incubation with goat anti-rabbit or goat anti-mouse HRP-conjugated secondary antibodies (1:4000; Abbkine) for 1 h at RT.

Techniques: Injection

Journal: Frontiers in Neuroscience

Article Title: Neuregulin1-ErbB4 Signaling in Spinal Cord Participates in Electroacupuncture Analgesia in Inflammatory Pain

doi: 10.3389/fnins.2021.636348

Figure Lengend Snippet: ErbB4 receptor is required for the effect of EA on CFA-induced hyperalgesia. (A) Experimental design of EA treatment, intrathecal injection of NRG1 or AG1478 in CFA-treated mice. Pain-related behaviors were tested from day –1 to day 7. (B,C) The mechanical threshold (B) and thermal latency (C) in mice treated with Saline, CFA + vehicle, CFA + EA, CFA + AG1478, and CFA + AG1478 + EA group. * p < 0.05, ** p < 0.01 vs. the CFA + vehicle group; # p < 0.05, ## p < 0.01 vs. the CFA + AG1478 + EA group. (D,E) The mechanical threshold (D) and thermal latency (E) in mice treated with Saline, CFA + vehicle, CFA + EA, CFA + NRG1 and CFA + NRG1 + EA group. * p < 0.05, ** p < 0.01 vs. the CFA + vehicle group. (B–E) : n = 10 mice per group. Data are presented as means ± SEM.

Article Snippet: The membranes were blocked with 5% non-fat milk for 1 h at RT before being probed with the primary antibodies: rabbit anti-ErbB4 (1:1000, Cell Signaling Technology, United States), mouse anti- ErbB4 (1: 500, Santa Cruz Biotechnology, United States), or rabbit anti-Neuregulin-1 (1: 500, Santa Cruz Biotechnology, United States) at 4°C overnight followed by incubation with goat anti-rabbit or goat anti-mouse HRP-conjugated secondary antibodies (1:4000; Abbkine) for 1 h at RT.

Techniques: Injection

Journal: Frontiers in Neuroscience

Article Title: Neuregulin1-ErbB4 Signaling in Spinal Cord Participates in Electroacupuncture Analgesia in Inflammatory Pain

doi: 10.3389/fnins.2021.636348

Figure Lengend Snippet: ErbB4 positive neurons are co-labeled with PV positive neurons in laminae III of spinal cord. (A) Breeding diagram for the generation of Erbb4-CreER; Rosa:LSL-tdTomato (Erbb4-td) mice (left). Experimental procedures of Tamoxifen injection after week 8 for 5 consecutive days and immunofluorescence analysis since week 10 (right). (B) The distribution of ErbB4 positive neurons (red) in the spinal cord (left) and DRG (right). Scale bar, 100 μm. (C) Spinal sections from Erbb4-td mice showing tdTomato signals (red) and CGRP, IB4 or PKC-γ (green). Scale bar, 100 μm. (D) Overlapping of ErbB4 (red) and VGAT (green). Scale bar, 100 μm. (E) Double labeling of ErbB4 (red) and PV (green) in laminae III. Left: scale bar, 50 μm. Right: scale bar, 10 μm. (F) The quantification of co-expression of ErbB4 and PKC-γ, VGAT or PV from spinal sections.

Article Snippet: The membranes were blocked with 5% non-fat milk for 1 h at RT before being probed with the primary antibodies: rabbit anti-ErbB4 (1:1000, Cell Signaling Technology, United States), mouse anti- ErbB4 (1: 500, Santa Cruz Biotechnology, United States), or rabbit anti-Neuregulin-1 (1: 500, Santa Cruz Biotechnology, United States) at 4°C overnight followed by incubation with goat anti-rabbit or goat anti-mouse HRP-conjugated secondary antibodies (1:4000; Abbkine) for 1 h at RT.

Techniques: Labeling, Injection, Immunofluorescence, Expressing

Journal: Frontiers in Neuroscience

Article Title: Neuregulin1-ErbB4 Signaling in Spinal Cord Participates in Electroacupuncture Analgesia in Inflammatory Pain

doi: 10.3389/fnins.2021.636348

Figure Lengend Snippet: Ablation of ErbB4 from PV neurons blocks the effect of NRG1 on CFA-induced mechanical hyperalgesia (A) Breeding diagram for the generation of PV-Cre; Erbb4 –/– (PV-Erbb4 –/– ) mice. (B) Scheme showing that rAAV-fPV-CRE-pAs was injected into spinal dorsal horn in Erbb4 f/f mice. (C) Experimental procedures of EA or NRG1 treatment in PV-Erbb4 –/– mice or AAV-PV-Erbb4 –/– mice and their control littermates. (D,E) The mechanical threshold (D) and thermal latency (E) in PV-Erbb4 –/– or AAV-PV-Erbb4 –/– mice and their controls. * p < 0.05, ** p < 0.01. (F,G) Changes of the mechanical threshold (F) and thermal latency (G) in Erbb4 f/f and PV-Erbb4 –/– mice treated with NRG1 or vehicle after CFA injection. (H,I) Changes of the mechanical threshold (H) and thermal latency (I) in Control and AAV-PV-Erbb4 –/– mice treated with NRG1 or vehicle after CFA injection. (D–I) : n = 10 mice per group. Data are presented as means ± SEM. (F–I) : * p < 0.05, ** p < 0.01 vs. the Erbb4 f/f + CFA + vehicle group or Control + CFA + vehicle group; # p < 0.05, ## p < 0.01 vs. the PV-Erbb4 –/– + CFA + NRG1 group or AAV-PV-Erbb4 –/– + CFA + NRG1 group;∈ p < 0.05, ∈∈ p < 0.01 vs. the PV-Erbb4 –/– + CFA + vehicle group or AAV-PV-Erbb4 –/– + CFA + vehicle group.

Article Snippet: The membranes were blocked with 5% non-fat milk for 1 h at RT before being probed with the primary antibodies: rabbit anti-ErbB4 (1:1000, Cell Signaling Technology, United States), mouse anti- ErbB4 (1: 500, Santa Cruz Biotechnology, United States), or rabbit anti-Neuregulin-1 (1: 500, Santa Cruz Biotechnology, United States) at 4°C overnight followed by incubation with goat anti-rabbit or goat anti-mouse HRP-conjugated secondary antibodies (1:4000; Abbkine) for 1 h at RT.

Techniques: Injection

Journal: iScience

Article Title: Profiling of ERBB receptors and downstream pathways reveals selectivity and hidden properties of ERBB4 antagonists

doi: 10.1016/j.isci.2024.108839

Figure Lengend Snippet: Selectivity of receptor activation and downstream signaling by single ligands correlate in barcoded assays (A) Heatmap showing stimulation profiles on ERBB receptors, HTR2A, and downstream signaling pathways. Assays for receptors were performed using barcoded split TEV, assays for signaling pathways with pathway sensors coupled to barcodes. Compound effects are shown as log2-transformed fold change. (B–H) Barcoded assays align with luciferase readouts. Visualization of selected data from (A), comparing barcoded assays (black) with luciferase assay readouts (red). Assays for receptors were performed using split TEV, assays for signaling pathways with pathway sensors. Dose response graphs for EGFR (B), ERBB4 (B), EGR1p only (D), and EGR1p and ERBB4 transfected (E), HTR2A (F), CRE and HTR2A transfected (G), and NFAT and HTR2A transfected (H) with single stimuli applied at increasing concentrations. EGFld, EGF-like domain. Error bars represent SEM, n = 3 for barcoded assays, and n = 6 for luciferase assays. See also Figure S2 and Table S2 .

Article Snippet: Rabbit monoclonal anti-HER4/ErbB4 (clone E200) , Abcam , Cat# ab32375; RRID: AB_731579.

Techniques: Activation Assay, Transformation Assay, Luciferase, Transfection

Journal: iScience

Article Title: Profiling of ERBB receptors and downstream pathways reveals selectivity and hidden properties of ERBB4 antagonists

doi: 10.1016/j.isci.2024.108839

Figure Lengend Snippet: The barcoded ERBBprofiler reveals known and previously uncharacterized selectivity properties of ERBB receptor antagonists (A) Heatmap showing antagonistic effects of compounds on ERBB receptors, HTR2A, and downstream signaling pathways in PC12 cells. Assays for receptors were performed using barcoded split TEV assays, assays for signaling pathways with barcoded pathway sensors. In addition to the increasing concentrations of the compounds shown, all assays contained constant concentrations of EGF (30 ng/mL), EGF-like domain (10 ng/mL), and serotonin (1 μM). Compound effects are shown as log2-transformed fold change. (B–E) Dose response graphs comparing drug selectivity for receptors EGFR and ERBB4 (B, D) and downstream MAPK signaling (C, E) of compounds AG1478 (B, C), and pyrotinib (D, E). Data was extracted from the heatmap shown in (A). n = 3. (F–I) Dose response graphs for CRE sensor responses in PC12 cells using luciferase as readout for AG1478 (F), osimertinib (G), poziotinib (H), and pyrotinib (I). In addition to the increasing concentrations of the compounds shown, all assays contained the constant stimulation mix as in (A). (J–M) Dose response graphs for calcium and cAMP assays using Fluo-4 a.m. and GloSensor, respectively, as readouts in PC12 cells treated with increasing concentrations of AG1478 (J), osimertinib (K), poziotinib (L), and pyrotinib (M). As in luciferase assays, the constant stimulation mix was constantly present, next to the mentioned compounds. Error bars represent SEM, n = 3 for barcode assays (B-E), n = 6 for luciferase, Fluo-4 a.m., and GloSensor assays (F-M). See also Figure S3 , Tables S3 and .

Article Snippet: Rabbit monoclonal anti-HER4/ErbB4 (clone E200) , Abcam , Cat# ab32375; RRID: AB_731579.

Techniques: Transformation Assay, Luciferase

Journal: iScience

Article Title: Profiling of ERBB receptors and downstream pathways reveals selectivity and hidden properties of ERBB4 antagonists

doi: 10.1016/j.isci.2024.108839

Figure Lengend Snippet: Pyrotinib reveals selectivity for ERBB4 over EGFR (A and B) Dose response assays comparing AG1478 selectivity for receptors EGFR and ERBB4 (A) and downstream MAPK signaling (B) using firefly luciferase assays in PC12 cells. Assays for receptors were performed using split TEV, assays for MAPK signaling were conducted with an EGR1p pathway sensor. In addition to the increasing concentrations of AG1478, EGFR and ERBB4 assays contained a constant concentration of EGF (30 ng/mL) or EGF-like domain (10 ng/mL), respectively. (C and D) Western blot analyses of p -EGFR (in A549 cells) (C) and p -ERBB4 (in T-47 cells) (D) using increasing concentrations of AG1478. (E and F) Quantification of (C) and (D). (G and H) Dose response assays comparing pyrotinib selectivity for receptors EGFR and ERBB4 (G) and downstream MAPK signaling (H) using firefly luciferase assays in PC12 cells. Assays were conducted as in (A, B). (I and J) Western blot analyses of p -EGFR (in A549 cells) (I) and p -ERBB4 (in T-47 cells) (J) using increasing concentrations of pyrotinib. (K and L) Quantification of (I) and (J). Error bars represent SEM, n = 6 for luciferase assays (A, B, G, H), n = 3 for Western blot assays (E, F, K, L).

Article Snippet: Rabbit monoclonal anti-HER4/ErbB4 (clone E200) , Abcam , Cat# ab32375; RRID: AB_731579.

Techniques: Luciferase, Concentration Assay, Western Blot

Journal: iScience

Article Title: Profiling of ERBB receptors and downstream pathways reveals selectivity and hidden properties of ERBB4 antagonists

doi: 10.1016/j.isci.2024.108839

Figure Lengend Snippet: The barcoded ERBBprofiler reveals novel ERBB4 selective antagonists (A) Heatmap showing antagonistic effects of LDC compounds on ERBB receptors, HTR2A, and downstream signaling pathways in PC12 cells. Assays for receptors were performed using barcoded split TEV assays, assays for signaling pathways using barcoded pathway sensors. In addition to the increasing concentrations of the compounds shown, all assays contained constant concentrations of EGF (30 ng/mL), EGF-like domain (10 ng/mL), and serotonin (1 μM). (B–E) Dose response graphs comparing drug selectivity for ERBB4 over EGFR (measured with split TEV) (B, C) and downstream MAPK signaling (measured with the EGR1p sensor) (D, E) of compound A (B, D), and compound B (C, E). Data was extracted from the heatmap shown in (A). (F–I) Orthogonal validation for compound B using Western blot analyses of p -EGFR and p -ERK1/2 (measured in A549 cells) (F) and p -ERBB4 and p -ERK1/2 (measured in T-47 cells) (G) using increasing concentrations of compound B (Cpd B). (H) Quantification of relative p -EGFR and p -ERBB4 from (F, G). (I) Quantification of relative p -ERK1/2 from (F, G). (J and K) In vitro kinase activity assays using LANCE assays for compound A (J) and compound B (K) showing dose response graphs comparing drug selectivity for ERBB4 (red) and EGFR (black). (L) Dose response graphs for CRE sensor responses in PC12 cells using luciferase as readout for compounds A (black) and B (red). In addition to the increasing concentrations of the compounds, the constant stimulation mix as in (A) was present. (M and N) Dose response graphs for calcium and cAMP assays using Fluo-4 a.m. (black) and GloSensor (red), respectively, as readouts in PC12 cells treated with increasing concentrations of compound A (M) and compound B (N). In addition to the compounds, the constant stimulation mix was present as in (A). Error bars represent SEM; n = 3 for barcode assays (B–E), Western blots (F–I) and in vitro kinase assays (J, K); n = 6 for luciferase assays, Fluo-4 a.m., and GloSensor assays (L–N). See also Figures S4 and .

Article Snippet: Rabbit monoclonal anti-HER4/ErbB4 (clone E200) , Abcam , Cat# ab32375; RRID: AB_731579.

Techniques: Western Blot, In Vitro, Activity Assay, Luciferase

Journal: iScience

Article Title: Profiling of ERBB receptors and downstream pathways reveals selectivity and hidden properties of ERBB4 antagonists

doi: 10.1016/j.isci.2024.108839

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-HER4/ErbB4 (clone E200) , Abcam , Cat# ab32375; RRID: AB_731579.

Techniques: Virus, Recombinant, Software