Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: HER3-Mediated Resistance to Hsp90 Inhibition Detected in Breast Cancer Xenografts by Affibody-based PET Imaging

doi: 10.1158/1078-0432.CCR-17-2754

Figure Lengend Snippet: (A) Scheme illustrating the radiolabeling reaction of DFO-conjugated ZHER3:8698 affibody molecule with the positron emitter 89Zr. (B) Saturation binding of 89Zr-DFO-ZHER3:8698 to MCF-7 cells. The data are expressed as the mean values ± SEM (n = 3 independent experiments). (C) HER3 expression in a panel of breast cancer cell lines. Representative Western blot from whole cell lysates, with GAPDH used as the loading control. (D) In vitro binding specificity of 89Zr-DFO-ZHER3:8698 in breast cancer cells and specific blocking using 100-fold molar excess of either unlabeled ZHER3:8698 or the natural HER3 ligand HRG. The data are expressed as the mean values ± SEM (n = 3 independent experiments). *P = 0.0357; *P = 0.0446; for MDA-MB468, **P = 0.0087; *P = 0.015 for MCF-7, and **P = 0.009; **P = 0.0097 for BT-474 cells.

Article Snippet: Immunoprecipitation Tissue lysates (300 μg) and whole cell lysates (200 μg) were incubated overnight in a thermomixer at 4°C and 650 rpm, with 10 μg of anti-HER3 mouse mAb (Merck Millipore, UK).

Techniques: Radioactivity, Binding Assay, Expressing, Western Blot, In Vitro, Blocking Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: HER3-Mediated Resistance to Hsp90 Inhibition Detected in Breast Cancer Xenografts by Affibody-based PET Imaging

doi: 10.1158/1078-0432.CCR-17-2754

Figure Lengend Snippet: (A) Representative 15 min coronal fused PET/CT images of mice bearing MCF-7, MDA-MB-468, or MDA-MB-231 xenografts. The mice received ~ 8 MBq of either 89Zr-DFO-ZHER3:8698 or 89Zr-DFO-ZTAQ via tail vein injection, with image acquisition taking place 3 h after injection. The arrowheads indicate the tumors and the kidneys. (B) Ex vivo biodistribution at 3 h after injection of the radioconjugates. Data are expressed as the mean values ± SD (n = 3 animals). *P = 0.0136; ***P = 0.0002; ****P < 0.0001. (C) Representative Western blot of whole tumor tissue lysates evaluating HER3, HER2 and EGFR expression in the indicated xenograft models. (D) Histopathological analysis of HER3 expression in MCF-7, MDA-MB-468, and MDA-MB-231 xenografts displaying the highest HER3 expression in MCF-7 xenografts and the lowest in MDA-MB-231. (E) Representative ex vivo autoradiography sections taken 3 h after injection of 89Zr-DFO-ZHER3:8698. (F) Autoradiography quantification from panel E as the intensity per region of interest area (A.U./cm2). Data are expressed as the mean values ± SD (n = 10 sections). **P = 0.0028; ****P < 0.0001. (G) Representative segmented xenografts following PET/CT image acquisition 3 h after 89Zr-DFO-ZHER3:8698 injection. These images highlight the greater radioactivity accumulation observed in MCF-7 xenografts, and the heterogeneity of uptake across the tumor burden. Color map defined within the tumor volume only.

Article Snippet: Immunoprecipitation Tissue lysates (300 μg) and whole cell lysates (200 μg) were incubated overnight in a thermomixer at 4°C and 650 rpm, with 10 μg of anti-HER3 mouse mAb (Merck Millipore, UK).

Techniques: Positron Emission Tomography-Computed Tomography, Injection, Ex Vivo, Western Blot, Expressing, Autoradiography, Radioactivity

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: HER3-Mediated Resistance to Hsp90 Inhibition Detected in Breast Cancer Xenografts by Affibody-based PET Imaging

doi: 10.1158/1078-0432.CCR-17-2754

Figure Lengend Snippet: MCF-7 xenografts were randomized into two groups: control and treatment. The treatment group received 40 mg/kg of AUY922 i.p. every second day for a period of two weeks. (A) Representative axial fused PET/CT images of mice bearing MCF-7 tumors. Each mouse received 7.2-8.1 MBq of 89Zr-DFO-ZHER3:8698 via tail vein injection, with image acquisition taking place at 3 h after injection. The mice were imaged before initiating AUY922 treatment (day 0), and following administration of the last treatment dose (day 14). The %ID/g ratios were determined by dividing the %ID/g on day 14 by that obtained on day 0. (B) Scatter plot of the %ID/g ratios for both control (n = 6) and AUY922-treated mice (n = 7). The horizontal lines indicate the mean for each group. *P = 0.0131. (C) Scatter plot of the ex vivo tumor biodistribution at 3 h after injection of the radioconjugate on day 14, for both control (n = 4) and AUY922-treated mice (n = 6). The horizontal lines indicate the mean per group. **P = 0.0036. (D) Histopathological analysis of control and AUY922-treated MCF-7 xenografts. Tumor sections were stained with hematoxylin and eosin (H&E), HER3, or CD31.

Article Snippet: Immunoprecipitation Tissue lysates (300 μg) and whole cell lysates (200 μg) were incubated overnight in a thermomixer at 4°C and 650 rpm, with 10 μg of anti-HER3 mouse mAb (Merck Millipore, UK).

Techniques: Positron Emission Tomography-Computed Tomography, Injection, Ex Vivo, Staining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: HER3-Mediated Resistance to Hsp90 Inhibition Detected in Breast Cancer Xenografts by Affibody-based PET Imaging

doi: 10.1158/1078-0432.CCR-17-2754

Figure Lengend Snippet: (A) HER receptors, IGF-1Rβ, and PI3K/AKT pathway activation were monitored by Western blot using whole tissue lysates from all control and AUY922-treated mice. Hsp70/72 expression was used as a surrogate for AUY922 treatment efficacy, and GAPDH as a loading control. (B) Minimum to maximum box & whiskers plot of the quantified protein expression represented in A. Data are expressed as the normalized protein expression per antibody for control and AUY922 groups. The black lines represent the median value. *P = 0.0306; **P = 0.0022; ****P < 0.0001. (C, D) Correlation between HER3/IGF-1Rβ and Hsp70/72 protein expression per mouse. The dashed lines represent the ninety-five percent confidence levels. (E) Correlation between %ID/g ratios obtained from 89Zr-DFO-ZHER3:8698 PET images and Hsp70/72 protein expression per mouse. The dashed lines represent the ninety-five percent confidence levels. (F) BT-474 and MCF-7 cells were treated with 32 nM of AUY922 for 48 h. Equal amounts of whole cell lysates were immunoprecipitated with a mouse anti-HER3 antibody followed by Western blot analysis of HER3 and IGF-1Rβ. Whole tissue lysates from control mouse C5 and AUY922-treated mouse A6 were also immunoprecipitated against HER3 and analyzed by Western blot. Ten percent of the input lysates were used as loading controls.

Article Snippet: Immunoprecipitation Tissue lysates (300 μg) and whole cell lysates (200 μg) were incubated overnight in a thermomixer at 4°C and 650 rpm, with 10 μg of anti-HER3 mouse mAb (Merck Millipore, UK).

Techniques: Activation Assay, Western Blot, Expressing, Immunoprecipitation

Journal: PLoS ONE

Article Title: Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

doi: 10.1371/journal.pone.0146100

Figure Lengend Snippet: (A) Cells expressing either ERBB4 (upper panel), ERBB4 and ERBB3 (middle panel) or ERBB3 receptors (lower panel) were cultured in medium without ligand, in the presence of 10 ng/ml IL3, or in the presence of 10 or 100 ng/ml NRG1. Cell numbers were counted at the indicated time points. (B) Parental Ba/F3 or Ba/F3 cells expressing ERBB3, ERBB4 or both receptors were incubated in the absence of IL3 and treated with 100 ng/mL NRG1 where indicated. Total cell lysate were then prepared and immunoblotted with phosphoepitope- and protein-specific antibodies for ERBB3, ERBB4, Akt and MEK.

Article Snippet: To measure ERBB3 cell surface expression, Ba/F3 cells expressing ERBB3 and ERBB4 and, as a control, cells expressing ERBB4 alone were analyzed on a FACSCanto flow cytometer (BD Biosciences) with an ERBB3 antibody conjugated to phycoerythrin (FAB3481P, R&D Systems).

Techniques: Expressing, Cell Culture, Incubation

Journal: PLoS ONE

Article Title: Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

doi: 10.1371/journal.pone.0146100

Figure Lengend Snippet: Ba/F3 ERBB3/ERBB4 or ERBB4 cells were treated in the three replicate experiments as indicated. Upon lysis and proteolytic digestion, peptides were differentially labeled with the three isotopic variant of mTRAQ and then pooled prior to peptide separation by high pH reversed phase chromatography and IMAC phosphopeptide enrichment. Phosphopeptide fractions were then analyzed by quantitative LC-MS on a LTQ Orbitrap Velos instrument. Lower panel: Characteristic mTRAQ patterns shown for a peptide harboring a NRG1-induced phosphosite in ERBB3/ERBB4 cells, which less strongly up-regulated in the absence of ERBB3 in ERBB4-expressing Ba/F3 cells.

Article Snippet: To measure ERBB3 cell surface expression, Ba/F3 cells expressing ERBB3 and ERBB4 and, as a control, cells expressing ERBB4 alone were analyzed on a FACSCanto flow cytometer (BD Biosciences) with an ERBB3 antibody conjugated to phycoerythrin (FAB3481P, R&D Systems).

Techniques: Lysis, Labeling, Variant Assay, Reversed-phase Chromatography, Phospho-proteomics, Liquid Chromatography with Mass Spectroscopy, Expressing

Journal: PLoS ONE

Article Title: Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

doi: 10.1371/journal.pone.0146100

Figure Lengend Snippet: (A) Volcano plot of NRG1-regulated phosphorylation in Ba/F3 cells expressing ERBB3 and ERBB4. (B) Volcano plot comparison of phosphorylation sites in NRG1-treated ERBB3/ERBB4 versus NRG1-treated ERBB4 expressing Ba/F3 cells. In both comparisons, log 10 -transformed, average phosphosite ratios are plotted against their standard deviations determined from mTRAQ replicate quantifications. Significantly regulated class I sites according to the Global Mean Rank test are depicted in red, all other sites in blue. The dashed grey lines indicate two-fold regulation.

Article Snippet: To measure ERBB3 cell surface expression, Ba/F3 cells expressing ERBB3 and ERBB4 and, as a control, cells expressing ERBB4 alone were analyzed on a FACSCanto flow cytometer (BD Biosciences) with an ERBB3 antibody conjugated to phycoerythrin (FAB3481P, R&D Systems).

Techniques: Phospho-proteomics, Expressing, Comparison, Transformation Assay

Journal: PLoS ONE

Article Title: Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

doi: 10.1371/journal.pone.0146100

Figure Lengend Snippet: Scatter plot of the mean ERBB3/ERBB4 ± NRG1 ratios with mean ERBB3/ERBB4 versus ERBB4 ratios from NRG1-treated cells. Reproducibly quantified ERBB3 phosphosites are encircled.

Article Snippet: To measure ERBB3 cell surface expression, Ba/F3 cells expressing ERBB3 and ERBB4 and, as a control, cells expressing ERBB4 alone were analyzed on a FACSCanto flow cytometer (BD Biosciences) with an ERBB3 antibody conjugated to phycoerythrin (FAB3481P, R&D Systems).

Techniques:

Journal: PLoS ONE

Article Title: Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

doi: 10.1371/journal.pone.0146100

Figure Lengend Snippet: Selected phosphorylation sites induced by NRG1 treatment.

Article Snippet: To measure ERBB3 cell surface expression, Ba/F3 cells expressing ERBB3 and ERBB4 and, as a control, cells expressing ERBB4 alone were analyzed on a FACSCanto flow cytometer (BD Biosciences) with an ERBB3 antibody conjugated to phycoerythrin (FAB3481P, R&D Systems).

Techniques: Phospho-proteomics, Sequencing

Journal: Journal of Thoracic Oncology

Article Title: Clinical Significance of Epidermal Growth Factor Receptors in Non-small Cell Lung Cancer and a Prognostic Role for HER2 Gene Copy Number in Female Patients

doi: 10.1097/jto.0b013e3181ea510a

Figure Lengend Snippet: FIGURE 1. Simplified schematic illustra- tion of Erb family members transducing signals to different oncogenes. *Pre- sumed nuclear translocation of membra- nous HER3.

Article Snippet: The antibodies used in the study were as follows: Phospho-EGF Receptor/EGFR (1:250; rabbit monoclonal, clone 53A5; #4407; Cell Signaling Technology, Danvers, MA); HER2/neu (prediluted by the manufacturer; rabbit monoclonal, clone 4B5; #790-100; Ventana Medical Systems, Illkirch, France); Nuclear nonphosphorylated HER3 receptor (Her3n; 1:250; mouse monoclonal, clone RTJ2; #NB100-2691; Novus Biologicals, Littleton, CO); Phospho-HER3/HER3 (1:250; rabbit monoclonal, clone 21D3; #4791; Cell Signaling Technology); HER4 (1:50; rabbit polyclonal, #RB-9045-R7; Thermo Fisher Scientific Anatomic Pathology, Fremont, CA).

Techniques: Translocation Assay

Journal: Molecular psychiatry

Article Title: APP and DYRK1A regulate axonal and synaptic vesicle protein networks and mediate Alzheimer's pathology in trisomy 21 neurons.

doi: 10.1038/s41380-022-01454-5

Figure Lengend Snippet: Fig. 3 Normalization of copy number of APP or DYRK1A, but not RCAN1 and SYNJ1, rescues elevated phosphorylated tau levels in T21 neurons. IPSC lines targeted at each of the candidate HSA21 loci were differentiated to neuronal fate in parallel to T21 iPSCs and analyzed at d21. Control T21 iNs in these comparisons had been exposed to the same CRISPR targeting pipeline as the targeted lines, but mutations at the targeted locus were not introduced. Three differentiation rounds were performed with three wells analyzed per genotype per differentiation. For each, media were collected for Aβ quantification via ELISA and cells lysed for WB at d21. Shown is a representative WB and quantifications of data collected for targeting at each locus: APP (a, b), DYRK1A (c, d), RCAN1 (e, f), and SYNJ1 (g, h). “Total tau” quantification is determined using the K9JA antibody. One-way ANOVA with Dunnett’s multiple comparison’s test, *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001.

Article Snippet: Antibodies for Immunocytochemistry and western blotting Antigen Host ICC/ WB Dilution Vendor Catalog # APP Mouse WB 1/1000 Millipore MAB348 DYRK1A Sheep WB 1/200 R&D Systems AF5407 RCAN1 Rabbit WB 1/200 Sigma D6694 SYNJ1 Rabbit WB 1/200 Sigma HPA011916 GAPDH Mouse WB 1/10000 Millipore MAB374 ACTB Mouse WB 1/5000 Sigma A5441 Tau (MAPT) Rabbit WB 1/2000 Dako A0024 (K9JA) P202,205 AT8 (MAPT) Mouse WB 1/250 ThermoFisher MN1020 EEA1 Rabbit ICC 1/100 CST 3288S βIII tubulin Mouse ICC 1/100 Millipore MAB1637 NEFH Rabbit ICC 1/80 SigmaAldrich N4142 POU3F2 Rabbit ICC 1/100 Cell Signaling Technologies 12137S

Techniques: Control, CRISPR, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Signals from the brain and olfactory epithelium control shaping of the mammalian nasal capsule cartilage

doi: 10.7554/eLife.34465

Figure Lengend Snippet:

Article Snippet: Antibody , ERBB3 , RnD Systems, AF4518 , , one to 500 in PBS-T over night at RT.

Techniques: Software, RNAscope, Hybridization