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egfr (tyr1173) (21a9) #4757 antibody (Cell Signaling Technology Inc)

Name: phospho-her4/erbb4 (tyr1284)/egfr (tyr1173) (21a9) #4757 antibody
Brand: Cell Signaling Technology Inc
Rating: 90 (1 reviews)

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Cell Signaling Technology Inc phospho-her4/erbb4 (tyr1284)/egfr (tyr1173) (21a9) #4757 antibody
Phospho Her4/Erbb4 (Tyr1284)/Egfr (Tyr1173) (21a9) #4757 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/phospho-her4/erbb4 (tyr1284)/egfr (tyr1173) (21a9) #4757 antibody/product/Cell Signaling Technology Inc
Average 90 stars, based on 1 article reviews

phospho-her4/erbb4 (tyr1284)/egfr (tyr1173) (21a9) #4757 antibody - by Bioz Stars, 2026-03

90/100 stars

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The effects of ErbB4 receptor agonist E4A and melatonin on cognitive and spatial memory deficits in D-gal-induced aging mice. (A) Representative swimming trajectories of mice subjected to different treatments in the Morris Water Maze (MWM) spatial probe test. (B) Escape latency across the five experimental groups. (C) Number of target crossings during the MWM probe test. (D) Time spent in the target quadrant during the MWM probe test. (E) Representative heat map showing interaction frequency with novel versus familiar objects. (F) Recognition index in the Novel Object Recognition test among the experimental groups. (G) Spontaneous alternation behaviors in the Y-maze test were assessed. Data were presented as mean ± SD (n = 6–8). Statistical significance was denoted as follows: *p < 0.05, **p < 0.01, ****p < 0.0001.

Journal: Frontiers in Pharmacology

Article Title: Targeted ErbB4 receptor activation prevents D-galactose-induced neuronal senescence via inhibiting ferroptosis pathway

doi: 10.3389/fphar.2025.1528604

Figure Lengend Snippet: The effects of ErbB4 receptor agonist E4A and melatonin on cognitive and spatial memory deficits in D-gal-induced aging mice. (A) Representative swimming trajectories of mice subjected to different treatments in the Morris Water Maze (MWM) spatial probe test. (B) Escape latency across the five experimental groups. (C) Number of target crossings during the MWM probe test. (D) Time spent in the target quadrant during the MWM probe test. (E) Representative heat map showing interaction frequency with novel versus familiar objects. (F) Recognition index in the Novel Object Recognition test among the experimental groups. (G) Spontaneous alternation behaviors in the Y-maze test were assessed. Data were presented as mean ± SD (n = 6–8). Statistical significance was denoted as follows: *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: The antibodies used, diluted in primary antibody diluents (P0023A, Beyotime), included: rabbit anti-phospho-ErbB4 (bs-3220R, Bioss; 1:1,000), rabbit anti-ErbB4 (AF6807, Beyotime; 1:1,000), rabbit anti-phospho-Akt1 (AF1546, Beyotime; 1:1,000), mouse anti-Akt1 (sc-377457, Santa Cruz Biotech; 1:1,000), rabbit anti-Nrf2 (AF7623, Beyotime; 1:1,000), rabbit anti-TFRC (AF8136, Beyotime; 1:1,000), rabbit anti-SLC7A11 (GB115276, Servicebio Biotech; 1:1,000), rabbit anti-GPX4 (AF7020, Beyotime; 1:1,000), rabbit anti-Lamin B1 (12987-1-AP, Proteintech, 1:5,000), rabbit anti-p53 (AF5258, Beyotime; 1:1,000), mouse anti-p21 (AP021, Beyotime; 1:1,000), rabbit anti-p16 (bs-23797R, Bioss Biotech, Beijing, China; 1:1,000), and rabbit anti-GAPDH (GB15002, Servicebio Biotech; 1:2000).

Techniques:

Effect of ErbB4 receptor agonist and melatonin can ameliorate D-gal-induced aging in hippocampus in mice. (A) Western blot analysis of Lamin B1, p53, p21, p16, GFAP, and Iba-1 protein expression levels in the hippocampus of mice. (B–G) Quantification of Lamin B1, p53, p21, p16, GFAP, and Iba-1 protein levels. Data are presented as mean ± SD, with n = 4–5. *p < 0.05, **p < 0.01, ***p < 0.001.

Journal: Frontiers in Pharmacology

Article Title: Targeted ErbB4 receptor activation prevents D-galactose-induced neuronal senescence via inhibiting ferroptosis pathway

doi: 10.3389/fphar.2025.1528604

Figure Lengend Snippet: Effect of ErbB4 receptor agonist and melatonin can ameliorate D-gal-induced aging in hippocampus in mice. (A) Western blot analysis of Lamin B1, p53, p21, p16, GFAP, and Iba-1 protein expression levels in the hippocampus of mice. (B–G) Quantification of Lamin B1, p53, p21, p16, GFAP, and Iba-1 protein levels. Data are presented as mean ± SD, with n = 4–5. *p < 0.05, **p < 0.01, ***p < 0.001.

Techniques: Western Blot, Expressing

ErbB4 receptor agonist and melatonin inhibit ferroptosis in hippocampus in D-gal-induced aging mice. (A) Immunofluorescence analysis of GPX4-positive cells, SLC7A11-positive cells, TFRC-positive cells in the dentate gyrus (DG) and CA1 regions. Scale bar = 50 μm. (B) Western blot analysis of Nrf2, TFRC, SLC7A11, and GPX4 protein expression levels in the hippocampus of mice. (C–F) Quantification of Nrf2, TFRC, SLC7A11, and GPX4 protein levels. Data are presented as mean ± SD, with n = 4–5. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Journal: Frontiers in Pharmacology

Article Title: Targeted ErbB4 receptor activation prevents D-galactose-induced neuronal senescence via inhibiting ferroptosis pathway

doi: 10.3389/fphar.2025.1528604

Figure Lengend Snippet: ErbB4 receptor agonist and melatonin inhibit ferroptosis in hippocampus in D-gal-induced aging mice. (A) Immunofluorescence analysis of GPX4-positive cells, SLC7A11-positive cells, TFRC-positive cells in the dentate gyrus (DG) and CA1 regions. Scale bar = 50 μm. (B) Western blot analysis of Nrf2, TFRC, SLC7A11, and GPX4 protein expression levels in the hippocampus of mice. (C–F) Quantification of Nrf2, TFRC, SLC7A11, and GPX4 protein levels. Data are presented as mean ± SD, with n = 4–5. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Techniques: Immunofluorescence, Western Blot, Expressing

The mitigation of D-gal-induced ferroptosis in HT22 cells by ErbB4 receptor agonist and melatonin. (A) Cell viability post D-gal exposure, evaluated using the CCK-8 assay (n = 5). (B) Cell viability following co-treatment with D-gal and ErbB4 receptor agonist, assessed via the CCK-8 assay (n = 6). (C) Cell viability following co-treatment with D-gal and melatonin, determined by the CCK-8 assay (n = 6). (D) Visualization of cellular senescence through SA-β-gal staining. Scale bar = 100 μm. (E) Quantification of SA-β-gal staining intensities (n = 3). (F) Western blot analysis was conducted to assess the expression levels of senescence-associated markers and ferroptosis-related proteins in HT22 cells. (G–M) Quantification of Nrf2, TFRC, SLC7A11, GPX4, p53, p21, and p16 levels was performed, with normalization to GAPDH (n = 4–7). (N) Intracellular GSH levels (n = 4). (O) Intracellular MDA levels (n = 5). (P) Quantitative analysis of ROS levels was undertaken (n = 4). (Q) Intracellular ROS generation was measured using DCFH-DA, wherein ROS activity was reflected as green fluorescence intensity. Scale bar = 200 μm. Data are presented as mean ± SD. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Journal: Frontiers in Pharmacology

Article Title: Targeted ErbB4 receptor activation prevents D-galactose-induced neuronal senescence via inhibiting ferroptosis pathway

doi: 10.3389/fphar.2025.1528604

Figure Lengend Snippet: The mitigation of D-gal-induced ferroptosis in HT22 cells by ErbB4 receptor agonist and melatonin. (A) Cell viability post D-gal exposure, evaluated using the CCK-8 assay (n = 5). (B) Cell viability following co-treatment with D-gal and ErbB4 receptor agonist, assessed via the CCK-8 assay (n = 6). (C) Cell viability following co-treatment with D-gal and melatonin, determined by the CCK-8 assay (n = 6). (D) Visualization of cellular senescence through SA-β-gal staining. Scale bar = 100 μm. (E) Quantification of SA-β-gal staining intensities (n = 3). (F) Western blot analysis was conducted to assess the expression levels of senescence-associated markers and ferroptosis-related proteins in HT22 cells. (G–M) Quantification of Nrf2, TFRC, SLC7A11, GPX4, p53, p21, and p16 levels was performed, with normalization to GAPDH (n = 4–7). (N) Intracellular GSH levels (n = 4). (O) Intracellular MDA levels (n = 5). (P) Quantitative analysis of ROS levels was undertaken (n = 4). (Q) Intracellular ROS generation was measured using DCFH-DA, wherein ROS activity was reflected as green fluorescence intensity. Scale bar = 200 μm. Data are presented as mean ± SD. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Techniques: CCK-8 Assay, Staining, Western Blot, Expressing, Activity Assay, Fluorescence

The effects of ErbB4 receptor agonist and melatonin treatment on ferroptosis in Erastin-exposed HT22 cells. (A) Cell viability of HT22 cells post-Erastin exposure was evaluated using the CCK-8 assay (n = 5). (B) Cell viability was evaluated following treatment with Erastin, cotreatment with either E4A or melatonin, or both, utilizing the CCK-8 assay (n = 4). (C) Western blot analysis was conducted to determine the expression levels of senescence-associated markers and ferroptosis-related proteins in HT22 cells. (D–I) Quantitative analysis of Nrf2, TFRC, SLC7A11, GPX4, p21, and p16 levels, normalized to GAPDH (n = 4–7). (J) Intracellular ROS generation was measured using DCFH-DA, with ROS activity indicated by green fluorescence. Scale bar = 200 μm. (K) The quantitative analysis of ROS levels was conducted (n = 4). The data are presented as mean ± SD. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Journal: Frontiers in Pharmacology

Article Title: Targeted ErbB4 receptor activation prevents D-galactose-induced neuronal senescence via inhibiting ferroptosis pathway

doi: 10.3389/fphar.2025.1528604

Figure Lengend Snippet: The effects of ErbB4 receptor agonist and melatonin treatment on ferroptosis in Erastin-exposed HT22 cells. (A) Cell viability of HT22 cells post-Erastin exposure was evaluated using the CCK-8 assay (n = 5). (B) Cell viability was evaluated following treatment with Erastin, cotreatment with either E4A or melatonin, or both, utilizing the CCK-8 assay (n = 4). (C) Western blot analysis was conducted to determine the expression levels of senescence-associated markers and ferroptosis-related proteins in HT22 cells. (D–I) Quantitative analysis of Nrf2, TFRC, SLC7A11, GPX4, p21, and p16 levels, normalized to GAPDH (n = 4–7). (J) Intracellular ROS generation was measured using DCFH-DA, with ROS activity indicated by green fluorescence. Scale bar = 200 μm. (K) The quantitative analysis of ROS levels was conducted (n = 4). The data are presented as mean ± SD. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Techniques: CCK-8 Assay, Western Blot, Expressing, Activity Assay, Fluorescence

ErbB4 receptor agonist mitigates D-gal-induced hippocampal neuronal aging both in vivo and in vitro through the activation of ErbB4 receptors and modulation of the Akt/Nrf2 signaling pathway. (A) Western blot analysis of pErbB4, ErbB4, pAkt1, and Akt1 protein expression levels in the hippocampus of mice. (B, C) Quantification of pErbB4 and pAkt1 levels (n = 4). (D) Western blot analysis of pErbB4, ErbB4, pAkt1, and Akt1 protein expression levels in D-gal-induced HT22 cells. (E, F) Quantification of pErbB4 and pAkt1 levels (n = 4). (G, H) Immunofluorescence analysis of the Nrf2-positive cells in the DG region (G) and in the cortex region (H) . Scale bar = 50 μm. (I) Western blot analysis of Nrf2, SLC7A11, GPX4, Lamin B1 and P21 protein levels after treatment of Nrf2 inhibitor AEM1. (J–N) Quantitative analysis of Nrf2, SLC7A11, GPX4, Lamin B1 and P21 levels (n = 4–7). Data are presented as mean ± SD. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Journal: Frontiers in Pharmacology

Article Title: Targeted ErbB4 receptor activation prevents D-galactose-induced neuronal senescence via inhibiting ferroptosis pathway

doi: 10.3389/fphar.2025.1528604

Figure Lengend Snippet: ErbB4 receptor agonist mitigates D-gal-induced hippocampal neuronal aging both in vivo and in vitro through the activation of ErbB4 receptors and modulation of the Akt/Nrf2 signaling pathway. (A) Western blot analysis of pErbB4, ErbB4, pAkt1, and Akt1 protein expression levels in the hippocampus of mice. (B, C) Quantification of pErbB4 and pAkt1 levels (n = 4). (D) Western blot analysis of pErbB4, ErbB4, pAkt1, and Akt1 protein expression levels in D-gal-induced HT22 cells. (E, F) Quantification of pErbB4 and pAkt1 levels (n = 4). (G, H) Immunofluorescence analysis of the Nrf2-positive cells in the DG region (G) and in the cortex region (H) . Scale bar = 50 μm. (I) Western blot analysis of Nrf2, SLC7A11, GPX4, Lamin B1 and P21 protein levels after treatment of Nrf2 inhibitor AEM1. (J–N) Quantitative analysis of Nrf2, SLC7A11, GPX4, Lamin B1 and P21 levels (n = 4–7). Data are presented as mean ± SD. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Techniques: In Vivo, In Vitro, Activation Assay, Western Blot, Expressing, Immunofluorescence

Schematic diagram illustrating the effects of targeted activation of ErbB4 receptor on neuronal aging via ferroptosis inhibition. Small molecule agonist (E4A)can activate ErbB4 receptors and regulate the Akt/Nrf2 signaling pathway to achieve neuroprotective effect in D-gal-induced neuronal aging. D-gal treatment increases the accumulation of advanced glycation end products (AGEs) which stimulates the production of reactive oxygen species (ROS) and increase the expression of senescence marker genes P53, P16, and P21. E4A can promote Akt1 phosphorylation and promote Nrf2 entrance into the nucleus, in which it involves in regulating the expression of ferroptosis suppressor gene, such as SLC7A11 and GPX4 to attenuate lipid peroxidation, which can inhibit cell ferroptosis and neuronal aging. The diagram was created with MedPeer ( www.medpeer.cn ).

Journal: Frontiers in Pharmacology

Article Title: Targeted ErbB4 receptor activation prevents D-galactose-induced neuronal senescence via inhibiting ferroptosis pathway

doi: 10.3389/fphar.2025.1528604

Figure Lengend Snippet: Schematic diagram illustrating the effects of targeted activation of ErbB4 receptor on neuronal aging via ferroptosis inhibition. Small molecule agonist (E4A)can activate ErbB4 receptors and regulate the Akt/Nrf2 signaling pathway to achieve neuroprotective effect in D-gal-induced neuronal aging. D-gal treatment increases the accumulation of advanced glycation end products (AGEs) which stimulates the production of reactive oxygen species (ROS) and increase the expression of senescence marker genes P53, P16, and P21. E4A can promote Akt1 phosphorylation and promote Nrf2 entrance into the nucleus, in which it involves in regulating the expression of ferroptosis suppressor gene, such as SLC7A11 and GPX4 to attenuate lipid peroxidation, which can inhibit cell ferroptosis and neuronal aging. The diagram was created with MedPeer ( www.medpeer.cn ).

Techniques: Activation Assay, Inhibition, Expressing, Marker

(a) Illustrative images depicting the expression levels of ErbB1, ErbB2, ErbB4, and β ‐actin. (b) Levels of ErbB1, ErbB2, and ErbB4 normalized to β ‐actin. (c) Gene expression levels of ErbB1, ErbB2, and ErbB4 measured by qPCR. Data are presented as mean ± SD ( n ≥ 3). # p < .1, ## p < .1, and #### p < .0001 compared with the control group, * p < .1, ** p < .01, *** p < .001, and **** p < .0001 compared with the heparin group.

Journal: Food Science & Nutrition

Article Title: Isothiocyanates attenuate heparin‐induced proliferation of colon cancer cells in vitro

doi: 10.1002/fsn3.4296

Figure Lengend Snippet: (a) Illustrative images depicting the expression levels of ErbB1, ErbB2, ErbB4, and β ‐actin. (b) Levels of ErbB1, ErbB2, and ErbB4 normalized to β ‐actin. (c) Gene expression levels of ErbB1, ErbB2, and ErbB4 measured by qPCR. Data are presented as mean ± SD ( n ≥ 3). # p < .1, ## p < .1, and #### p < .0001 compared with the control group, * p < .1, ** p < .01, *** p < .001, and **** p < .0001 compared with the heparin group.

Article Snippet: Protein bands were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA) and blocked with 5% nonfat milk in TBST (Tris‐buffered saline containing 0.1% Tween 20) at room temperature for 2 h. The membranes were incubated overnight with primary antibodies at 4°C targeting TGF‐ β (CST, 3709S), EGFR (ErbB1) (epidermal growth factor receptor) (CST, 4267T), HER2 (ErbB2) (receptor tyrosine‐protein kinase erbB‐2) (CST, 4290T), HER3 (ErbB3) (receptor tyrosine‐protein kinase erbB‐3) (CST, 12708T), HER4 (ErbB4) (receptor tyrosine‐protein kinase erbB‐4) (CST, 4795T), Bcl‐2 (B‐cell lymphoma 2) (CST, 3498T), Bax (Bcl‐2‐associated X protein) (CST, 2772T), and β ‐actin (CST, 8457T).

Techniques: Expressing, Gene Expression, Control

Surface Expression of Various Growth Factor Receptors in Human Brain Cancer Cell Line (Determined by Flow Cytometry)

Journal: World Journal of Oncology

Article Title: Synergistic Effects of Neratinib in Combination With Palbociclib or Miransertib in Brain Cancer Cells

doi: 10.14740/wjon1873

Figure Lengend Snippet: Surface Expression of Various Growth Factor Receptors in Human Brain Cancer Cell Line (Determined by Flow Cytometry)

Article Snippet: Other antibodies for the Western blot analysis, such as the rabbit anti-phospho-EGFR (Tyr1068), HER2 (2242s), phosphor-HER2 (Tyr1221/1222) (2243), phosphor-HER3 (Tyr1289) (4791), phosphor-HER4 (Tyr1284)/EGFR (Tyr1173) (4757), phosphor-MAPK (Tyr202/Tyr204) (4370), phospho-Akt (S473) (4060), phospho-STAT3 (Y705) (9145), phospho-SRC (Y416) (Tyr416) (6942) and β-actin (4970), were all obtained from Cell Signaling Technology Inc. (Hitchin, UK).

Techniques: Expressing, Cytometry, Fluorescence

IC 50 Values of Various Agents on HBCCLs as Assessed by SRB Colorimetric Assay: (A) HER-Family Targeting TKIs and Other Downstream Signaling Molecules and (B) Other TKIs and Chemotherapeutic Agents

Journal: World Journal of Oncology

Article Title: Synergistic Effects of Neratinib in Combination With Palbociclib or Miransertib in Brain Cancer Cells

doi: 10.14740/wjon1873

Figure Lengend Snippet: IC 50 Values of Various Agents on HBCCLs as Assessed by SRB Colorimetric Assay: (A) HER-Family Targeting TKIs and Other Downstream Signaling Molecules and (B) Other TKIs and Chemotherapeutic Agents

Techniques: Colorimetric Assay, Polymer

( a ) Cartoon schematic of the HER2/HER4/NRG1β heterodimer depicts the assembly of a ‘heart-shaped’ ectodomain dimer upon binding of a ligand/growth factor (GF) to HER4. Individual domains of the HER ectodomains are annotated as domain ( D ) I – IV. The intracellular kinase domains assemble into an asymmetric dimer in which HER2 adopts the receiver (activated) and HER4 the activator (inactive) positions, enforced by the interface mutations: HER2-V956R and HER4-I712Q, respectively. ( b–c ) Structures of the near full-length HER2-V956R/HER4-I712Q complex (labeled HER2/HER4) bound to NRG1β or BTC. The ectodomain models are shown in cartoon representation fitted into the cryo-EM density. Only density for the ectodomain modules was observed. Domains I-IV are labeled DI-DIV. ( d ) Overlay of HER2/HER4 heterodimers bound to NRG1β and BTC aligned on the HER2 chain (RMSD 0.835 Å). ( e ) 3D classification analysis of HER2/HER4 heterodimers bound to NRG1β or BTC. Overlay of models in ribbon resulting from the 3D classification of particles into four classes are shown (HER2/HER4/NRG1β 289,192 particles, HER2/HER4/BTC 148,541 particles). Models were aligned on the HER2 chain.

Journal: eLife

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.7554/eLife.92873

Figure Lengend Snippet: ( a ) Cartoon schematic of the HER2/HER4/NRG1β heterodimer depicts the assembly of a ‘heart-shaped’ ectodomain dimer upon binding of a ligand/growth factor (GF) to HER4. Individual domains of the HER ectodomains are annotated as domain ( D ) I – IV. The intracellular kinase domains assemble into an asymmetric dimer in which HER2 adopts the receiver (activated) and HER4 the activator (inactive) positions, enforced by the interface mutations: HER2-V956R and HER4-I712Q, respectively. ( b–c ) Structures of the near full-length HER2-V956R/HER4-I712Q complex (labeled HER2/HER4) bound to NRG1β or BTC. The ectodomain models are shown in cartoon representation fitted into the cryo-EM density. Only density for the ectodomain modules was observed. Domains I-IV are labeled DI-DIV. ( d ) Overlay of HER2/HER4 heterodimers bound to NRG1β and BTC aligned on the HER2 chain (RMSD 0.835 Å). ( e ) 3D classification analysis of HER2/HER4 heterodimers bound to NRG1β or BTC. Overlay of models in ribbon resulting from the 3D classification of particles into four classes are shown (HER2/HER4/NRG1β 289,192 particles, HER2/HER4/BTC 148,541 particles). Models were aligned on the HER2 chain.

Article Snippet: HER2, HER4, phospho-HER2 (pY1221/1222) and pHER4 (1284) levels were determined by Western blot using following antibodies: rabbit anti-HER4 (Cell Signaling, Cat# 111B2, 1:1000, RRID: AB_2099883 ), rabbit anti-phospho-Y1283 HER4 (Cell Signaling, Cat# 21A9, 1:1000, RRID: AB_2099987 ), rabbit anti-HER2 (Cell Signaling, Cat# D8F12, 1:1000, RRID: AB_10557104 ), rabbit anti-phospho-Y1221/1222 HER2 (Cell Signaling, Cat# 2249, 1:1000, RRID: AB_2099241 ), anti-rabbit IgG HRP-linked antibody (Cell Signaling, Cat# 7074, 1:5000, RRID: AB_2099233 ).

Techniques: Binding Assay, Labeling, Cryo-EM Sample Prep

( a ) Cryo-EM map at different contour levels. ( b ) CryoSPARC GSFSC plots. ( c ) CryoSPARC Euler angle plots. ( d ) 3DFSC plots. ( e ) Model-Map-FSC curves from Phenix Validation ( f ) Local resolution map of HER2/HER4/NRG1βcreated using cryoSPARC v4.

Journal: eLife

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.7554/eLife.92873

Figure Lengend Snippet: ( a ) Cryo-EM map at different contour levels. ( b ) CryoSPARC GSFSC plots. ( c ) CryoSPARC Euler angle plots. ( d ) 3DFSC plots. ( e ) Model-Map-FSC curves from Phenix Validation ( f ) Local resolution map of HER2/HER4/NRG1βcreated using cryoSPARC v4.

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.7554/eLife.92873

Techniques: Cryo-EM Sample Prep

Cryo-EM data collection, refinement, and validation statistics.

Journal: eLife

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.7554/eLife.92873

Figure Lengend Snippet: Cryo-EM data collection, refinement, and validation statistics.

Techniques:

( a ) Overlays of indicated homo- and heterodimers. Heterodimer alignments were performed using the HER2 chain, and alignments with HER4 homodimers were performed using the HER4 chain. The dotted line represents a C2 symmetry axis highlighting the asymmetry of heterodimers compared to near-perfect C2 symmetry observed for HER4/NRG1β homodimers. ( b ) Individual receptors from the HER2-containing heterodimers were aligned using the HER2 chain or its co-receptor chain, as indicated. HER2-only is cryo-EM structure of the HER2 ECD with Pertuzumab and Trastuzumab Fab bound (PDB: 6OGE; Fabs not shown), HER2/HER3/NRG1β (PDB: 7MN5), HER2-S310F/HER3/NRG1β (PDB: 7MN6), HER2/EGFR/EGF (PDB: 8HGO). ( c ) Comparison between indicated HER2 heterodimer structures. Structural models are overlayed on HER2 to highlight nuances with which HER2 engages its co-receptors. The same PDB codes were used as in ( b ).

Journal: eLife

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.7554/eLife.92873

Figure Lengend Snippet: ( a ) Overlays of indicated homo- and heterodimers. Heterodimer alignments were performed using the HER2 chain, and alignments with HER4 homodimers were performed using the HER4 chain. The dotted line represents a C2 symmetry axis highlighting the asymmetry of heterodimers compared to near-perfect C2 symmetry observed for HER4/NRG1β homodimers. ( b ) Individual receptors from the HER2-containing heterodimers were aligned using the HER2 chain or its co-receptor chain, as indicated. HER2-only is cryo-EM structure of the HER2 ECD with Pertuzumab and Trastuzumab Fab bound (PDB: 6OGE; Fabs not shown), HER2/HER3/NRG1β (PDB: 7MN5), HER2-S310F/HER3/NRG1β (PDB: 7MN6), HER2/EGFR/EGF (PDB: 8HGO). ( c ) Comparison between indicated HER2 heterodimer structures. Structural models are overlayed on HER2 to highlight nuances with which HER2 engages its co-receptors. The same PDB codes were used as in ( b ).

Techniques: Cryo-EM Sample Prep, Comparison

( a ) Cryo-EM density and model of the HER2/HER4/NRG1β domain II at two different orientations highlight two equally well-resolved dimerization arms. ( b ) Hydrogen-bonds, cation-π interactions, and salt bridges are depicted at the dimer interface, with other residues omitted for clarity. The HER2 and HER4 dimerization arms engage in the same set of polar interactions (insets B and C), except for a cation-π interaction between HER2 F279 with HER4 R306 ( A ) due to a substitution of the equivalent of HER4 R306 to L313 in HER2. Residues labeled ‘DI’ are in receptor domain I while all others are in domain II (DII). Interface residues and hydrogen bonds were determined using UCSF ChimeraX. ( c ) Known HER2 heterodimers are aligned using the HER2 chain to highlight the positioning of the dimerization arms. ( d ) Dimerization arm regions of selected HER receptor dimers are shown colored by B-factors. B-factor colors were scaled to represent max and min B-factor values within each structure corresponding to different absolute values across structures due to variability in their resolution. Distance measurements at fixed points highlight a correlation between asymmetrically distributed B-factors and asymmetrically engaged dimerization arms. ( e ) Western blot analysis of NR6 cell lysates transduced with indicated HER2 and HER4 constructs. Cells were starved for 4 h prior to stimulation with 10 nM NRG1β at 37 °C for 10 min. Molecular weight markers (in kDa) are indicated next to each blot. Figure 2—source data 1. Original files for western blot analysis in . Figure 2—source data 2. Original files for western blot analysis in with all labels and cropped areas shown.

Journal: eLife

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.7554/eLife.92873

Figure Lengend Snippet: ( a ) Cryo-EM density and model of the HER2/HER4/NRG1β domain II at two different orientations highlight two equally well-resolved dimerization arms. ( b ) Hydrogen-bonds, cation-π interactions, and salt bridges are depicted at the dimer interface, with other residues omitted for clarity. The HER2 and HER4 dimerization arms engage in the same set of polar interactions (insets B and C), except for a cation-π interaction between HER2 F279 with HER4 R306 ( A ) due to a substitution of the equivalent of HER4 R306 to L313 in HER2. Residues labeled ‘DI’ are in receptor domain I while all others are in domain II (DII). Interface residues and hydrogen bonds were determined using UCSF ChimeraX. ( c ) Known HER2 heterodimers are aligned using the HER2 chain to highlight the positioning of the dimerization arms. ( d ) Dimerization arm regions of selected HER receptor dimers are shown colored by B-factors. B-factor colors were scaled to represent max and min B-factor values within each structure corresponding to different absolute values across structures due to variability in their resolution. Distance measurements at fixed points highlight a correlation between asymmetrically distributed B-factors and asymmetrically engaged dimerization arms. ( e ) Western blot analysis of NR6 cell lysates transduced with indicated HER2 and HER4 constructs. Cells were starved for 4 h prior to stimulation with 10 nM NRG1β at 37 °C for 10 min. Molecular weight markers (in kDa) are indicated next to each blot. Figure 2—source data 1. Original files for western blot analysis in . Figure 2—source data 2. Original files for western blot analysis in with all labels and cropped areas shown.

Techniques: Cryo-EM Sample Prep, Labeling, Western Blot, Transduction, Construct, Molecular Weight

( a ) Sequence alignment of HER receptor dimerization arm regions with conserved residues highlighted in red. Two aromatic residues that are known to engage in hydrogen bonding with the partner receptor are marked with (*). ( b ) Full domains II ( DII ) for selected receptor dimers are shown in the cartoon and all the interface residues between two receptors within domains I and III ( DI-DIII ) are shown as sticks. Hydrogen bonds are indicated with dotted lines. Analysis was performed using UCSF ChimeraX. Domains IV are not resolved in most structures and are not included in this analysis. Canonical dimerization arm interactions involve domains DI and DII, while non-canonical interfaces, as seen for EGFR in the HER2/EGFR/EGF dimer and one EGFR/EREG monomer in the EGFR/EREG homodimer, engage DIII instead of DI. The buried surface area (BSA) at each interface is indicated. If more than one crystallographic dimer was observed in an asymmetric unit, the range of BSA values for all observed dimers is indicated. The following PDB codes were used: HER2/HER3/NRG1β (PDB: 7MN5), HER2/EGFR/EGF (PDB: 8HGO), EGFR/EGF (PDB: 3NJP), HER4/NRG1β (PDB: 3U7U), and EGFR/EREG (PDB: 5WB7).

Journal: eLife

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.7554/eLife.92873

Figure Lengend Snippet: ( a ) Sequence alignment of HER receptor dimerization arm regions with conserved residues highlighted in red. Two aromatic residues that are known to engage in hydrogen bonding with the partner receptor are marked with (*). ( b ) Full domains II ( DII ) for selected receptor dimers are shown in the cartoon and all the interface residues between two receptors within domains I and III ( DI-DIII ) are shown as sticks. Hydrogen bonds are indicated with dotted lines. Analysis was performed using UCSF ChimeraX. Domains IV are not resolved in most structures and are not included in this analysis. Canonical dimerization arm interactions involve domains DI and DII, while non-canonical interfaces, as seen for EGFR in the HER2/EGFR/EGF dimer and one EGFR/EREG monomer in the EGFR/EREG homodimer, engage DIII instead of DI. The buried surface area (BSA) at each interface is indicated. If more than one crystallographic dimer was observed in an asymmetric unit, the range of BSA values for all observed dimers is indicated. The following PDB codes were used: HER2/HER3/NRG1β (PDB: 7MN5), HER2/EGFR/EGF (PDB: 8HGO), EGFR/EGF (PDB: 3NJP), HER4/NRG1β (PDB: 3U7U), and EGFR/EREG (PDB: 5WB7).

Techniques: Sequencing

( a ) Structures of full-length HER4 homodimers bound to either NRG1β or BTC. Only density for the ectodomain modules was observed in both structures, shown here as a cartoon representation fitted into the cryo-EM density. ( b ) Comparison between the NRG1β− and BTC-bound HER4 dimers. Angle measurements were derived using UCSF ChimeraX by defining an axis through each receptor in a dimer and measuring the angle between the two axes. ( c ) Overlays of ribbon models obtained by 3D classification of particles into four distinct classes are shown for HER4 homodimers bound to NRG1β or BTC (205,726 particles HER4/NRG1β and ~274,540 particles HER4/BTC). Classification was performed in cryoSPARC using the heterogeneous refinement job with four identical start volumes and particles from final reconstructions are shown in ( a ). ( d-e ) Overlays of HER4 receptor homodimers bound to NRG1β or BTC show differences in the ligand binding pockets and how receptors assemble into dimers. Receptors were aligned as indicated in the panels. The HER4-NRG1β engages 4 salt bridges in the binding pocket, three of which are not present in HER4-BTC (shown in boxes). The salt bridge involving HER4 K35 can only be confidently observed in cryo-EM maps of one monomer (chain A).

Journal: eLife

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.7554/eLife.92873

Figure Lengend Snippet: ( a ) Structures of full-length HER4 homodimers bound to either NRG1β or BTC. Only density for the ectodomain modules was observed in both structures, shown here as a cartoon representation fitted into the cryo-EM density. ( b ) Comparison between the NRG1β− and BTC-bound HER4 dimers. Angle measurements were derived using UCSF ChimeraX by defining an axis through each receptor in a dimer and measuring the angle between the two axes. ( c ) Overlays of ribbon models obtained by 3D classification of particles into four distinct classes are shown for HER4 homodimers bound to NRG1β or BTC (205,726 particles HER4/NRG1β and ~274,540 particles HER4/BTC). Classification was performed in cryoSPARC using the heterogeneous refinement job with four identical start volumes and particles from final reconstructions are shown in ( a ). ( d-e ) Overlays of HER4 receptor homodimers bound to NRG1β or BTC show differences in the ligand binding pockets and how receptors assemble into dimers. Receptors were aligned as indicated in the panels. The HER4-NRG1β engages 4 salt bridges in the binding pocket, three of which are not present in HER4-BTC (shown in boxes). The salt bridge involving HER4 K35 can only be confidently observed in cryo-EM maps of one monomer (chain A).

Techniques: Cryo-EM Sample Prep, Comparison, Derivative Assay, Ligand Binding Assay, Binding Assay

Journal: eLife

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.7554/eLife.92873

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.7554/eLife.92873

Figure Lengend Snippet: ( a ) Cryo-EM map at different contour levels. ( b ) CryoSPARC GSFSC plots. ( c ) CryoSPARC Euler angle plots. ( d ) 3DFSC plots. ( e ) Model-Map-FSC curves from Phenix Validation. ( f ) Local resolution map of HER4/BTC created using cryoSPARC v4.

Techniques: Cryo-EM Sample Prep

( a ) Overlay of three HER4/NRG1β ectodomain homodimers found in the asymmetric unit of the crystal structure (PDB: 3U7U) with the cryo-EM structure of full-length HER4/NRG1β. The crystal structure models are shown in gray. RMSDs for overlay of full dimers with the cryo-EM HER4/NRG1β dimer are 5.438 Å, 5.435 Å, and 3.662 Å, respectively. ( b ) HER4/NRG1β model built into C1 refined cryo-EM map was aligned across chains (chain A in one model aligned to chain B in another model). While the aligned chain showed a near-perfect match (RMSD 1.42 Å), the other chain showed a breaking of C2 symmetry. ( c ) HER4/BTC model built into C1 refined cryo-EM map was aligned across chains (chain A in one model aligned to chain B in another model). While the aligned chain showed a near-perfect match (RMSD 1.58 Å), the other chain showed a breaking of C2 symmetry.

Journal: eLife

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.7554/eLife.92873

Figure Lengend Snippet: ( a ) Overlay of three HER4/NRG1β ectodomain homodimers found in the asymmetric unit of the crystal structure (PDB: 3U7U) with the cryo-EM structure of full-length HER4/NRG1β. The crystal structure models are shown in gray. RMSDs for overlay of full dimers with the cryo-EM HER4/NRG1β dimer are 5.438 Å, 5.435 Å, and 3.662 Å, respectively. ( b ) HER4/NRG1β model built into C1 refined cryo-EM map was aligned across chains (chain A in one model aligned to chain B in another model). While the aligned chain showed a near-perfect match (RMSD 1.42 Å), the other chain showed a breaking of C2 symmetry. ( c ) HER4/BTC model built into C1 refined cryo-EM map was aligned across chains (chain A in one model aligned to chain B in another model). While the aligned chain showed a near-perfect match (RMSD 1.58 Å), the other chain showed a breaking of C2 symmetry.

Techniques: Cryo-EM Sample Prep

( a ) Model of the HER4/NRG1β homodimer fitted into the cryo-EM density, lowpass-filtered to 6 Å, reveals multiple glycans that mediate intra- and interreceptor connections. Glycans are shown in blue. Insets ( A and B ) are close-up views of glycans connected to N138 and N253, and are shown at a higher volume contour than the central heterodimer. Insets ( C and D ) are close-up views of glycans connected to N548, N576, and N358. ( D ) shows continuous glycan density originating from N576 of one receptor and connecting to N358 of the dimerization partner. Maps are shown at lower contour than in the central heterodimer. Various contour levels are shown in for reference. Arrows indicate regions in which the cryo-EM map from one glycan merges with the density of glycans or polypeptide chains from different HER receptor sub-domains. ( b ) Model of HER2/HER4/NRG1β fitted into cryo-EM density, lowpass-filtered to 6 Å, reveals intra-receptor glycosylation only. Insets ( A ) shows HER4 glycosylation on N548 and N576 pointing from HER4 domain IV to domain II, but less pronounced as observed in HER4 homodimers. Glycan connections between domain I and II in HER4, via N138 and N253-linked glycans, are comparable to the ones seen in HER4 homodimer shown in inset ( A ). Inset ( B ) shows the equivalent glycan connections in domain I and II of HER2. Inset ( C ) reveals missing glycosylation sites at equivalent positions in HER2; G366, N556, and Q583 (pink).

Journal: eLife

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.7554/eLife.92873

Figure Lengend Snippet: ( a ) Model of the HER4/NRG1β homodimer fitted into the cryo-EM density, lowpass-filtered to 6 Å, reveals multiple glycans that mediate intra- and interreceptor connections. Glycans are shown in blue. Insets ( A and B ) are close-up views of glycans connected to N138 and N253, and are shown at a higher volume contour than the central heterodimer. Insets ( C and D ) are close-up views of glycans connected to N548, N576, and N358. ( D ) shows continuous glycan density originating from N576 of one receptor and connecting to N358 of the dimerization partner. Maps are shown at lower contour than in the central heterodimer. Various contour levels are shown in for reference. Arrows indicate regions in which the cryo-EM map from one glycan merges with the density of glycans or polypeptide chains from different HER receptor sub-domains. ( b ) Model of HER2/HER4/NRG1β fitted into cryo-EM density, lowpass-filtered to 6 Å, reveals intra-receptor glycosylation only. Insets ( A ) shows HER4 glycosylation on N548 and N576 pointing from HER4 domain IV to domain II, but less pronounced as observed in HER4 homodimers. Glycan connections between domain I and II in HER4, via N138 and N253-linked glycans, are comparable to the ones seen in HER4 homodimer shown in inset ( A ). Inset ( B ) shows the equivalent glycan connections in domain I and II of HER2. Inset ( C ) reveals missing glycosylation sites at equivalent positions in HER2; G366, N556, and Q583 (pink).

Techniques: Cryo-EM Sample Prep

( a ) Model of the HER4/NRG1β homodimer fitted into the cryo-EM density, lowpass-filtered to 6 Å, at various contour levels. Glycans are shown in blue. ( b ) 3D classification of the HER4/NRG1β particles reveals strong continuous glycan density between two receptors within the dimer for class 3. ( c ) Glycosylation site asparagines in EGFR, HER2, HER3, and HER4 are marked in blue, and shown in sphere representation. Glycosylation site asparagines involved in inter-receptor contacts in our HER4 structures, and the equivalent residues in other HER receptors are indicated by teal labels. ( d ) Analysis of the cryo-EM map of the EGFR/EGF homodimer structure (PDB: 7SYD) at various contour levels suggests the presence of an inter-receptor glycan connection between N353 and N603, shown in blue.

Journal: eLife

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.7554/eLife.92873

Figure Lengend Snippet: ( a ) Model of the HER4/NRG1β homodimer fitted into the cryo-EM density, lowpass-filtered to 6 Å, at various contour levels. Glycans are shown in blue. ( b ) 3D classification of the HER4/NRG1β particles reveals strong continuous glycan density between two receptors within the dimer for class 3. ( c ) Glycosylation site asparagines in EGFR, HER2, HER3, and HER4 are marked in blue, and shown in sphere representation. Glycosylation site asparagines involved in inter-receptor contacts in our HER4 structures, and the equivalent residues in other HER receptors are indicated by teal labels. ( d ) Analysis of the cryo-EM map of the EGFR/EGF homodimer structure (PDB: 7SYD) at various contour levels suggests the presence of an inter-receptor glycan connection between N353 and N603, shown in blue.

Techniques: Cryo-EM Sample Prep

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