rabbit antihuman erbb3  (Santa Cruz Biotechnology)

Journal: Science (New York, N.Y.)

Article Title: A protein network map of head and neck cancer reveals PIK3CA mutant drug sensitivity

doi: 10.1126/science.abf2911

Figure Lengend Snippet: (A) Overview of the PIK3CA signaling pathway, which is often stimulated by RTKs that interact with PIK3CA to stimulate RAS/Raf-mediated or Akt/mTORC1-mediated downstream signaling. (B) Analyzed PIK3CA mutants and their frequency in HNSCC tumors from TCGA. Asterisk (*) denotes mutations annotated as oncogenic in OncoKB (65). Graph bars corresponding to each mutation were color-coded to indicate their localization within the p110α domain (as indicated in the legend in top right corner). (C) Selected PIK3CA mutations were mapped on the structure of PI3K (PDB: 4L23) (66) by highlighting the mutated residues as red spheres. (D) Quantification of PPIs for all PIK3CA HC-PPIs detected in the SCC-25 cell line (all cell lines displayed in Figure S4A). (E) Cartoon representation of a zoomed-in view of PI3K illustrating a salt bridge formed between K11 and E81 (PDB: 4L23). (F) A zoomed-in view depicting interactions made by G1007 in PI3K (PDB: 4L23). (G) Cartoon representation of different mutation induced PI3K activation mechanisms and their respective HER3 binding preferences.

Article Snippet: Gαi-DREADD (pcDNA3.1) Antibodies RSK1/2/3 antibody Cell Signaling Technology 9355 ERK1/2 Cell Signaling Technology 4695 phospho-PAK1(S199/204)/PAK2(S192/197) Cell Signaling Technology 2605 PAK1 Cell Signaling Technology 2602 PAK2 Cell Signaling Technology 2608 pERK Cell Signaling Technology 9106 FGFR3 OriGene TA801078 Daple Millipore EMD ABS515 GAPDH Cell Signaling Technology 2118 secondary goat anti-rabbit HRP Southern Biotech 4010-05 P-HER3-Y1197 Cell Signaling Technology 4561 HER3 Cell Signaling Technology 12708 goat anti-mouse HRP Southern Biotech 1010-05 anti-B-tubulin Abcam ab6276 ERK Cell Signaling Technology 9102 Deposited data Unprocessed peptide files This paper PRIDE ProteomeXchange: PXD019469 Raw data This paper PRIDE ProteomeXchange: PXD019469 Chemicals, Peptides, and Recombinant Proteins Tris G-Biosciences RC108 Acetonitrile, HPLC grade (ACN) Thermo Fisher Scientific A955-4 cOmplete protease inhibitor cocktail tablets mini, EDTA-free Roche 11846 170 001 Dithiothreitol (DTT) Sigma-Aldrich 43819 Formic acid (FA) Thermo Fisher Scientific 28905 Iodoacetamide (IAA) Acros Organic 122270250 Sequencing-grade modified trypsin Promega V5111 Benzonase Sigma E1014-25KU Trifluoroacetic acid (TFA) Thermo Fisher Scientific 28904 Urea Sigma-Aldrich U5378-1kg Fetal bovine serum (FBS) Gibco A3160502 DMEM Corning MT10013CV DMEM/F12 Corning MT10092CV Water, HPLC grade Sigma-Aldrich 270733-4 L Igepal (NP-40) Sigma-Aldrich I3021 Minimal Essential Media Corning 10-009-CV Opti-MEM Thermo Fisher Scientific 31985062 BEGM™ (Lonza) Lonza CC-3170 1% Penicillin-Streptomycin Corning MT30002Cl Paraformaldehyde, 4% solution in PBS Thermo Scientific MFCD00133991 PolyJet SignaGen SL100688 Lipofectamine 3000 ThermoFisher Scientific L3000008 hydrocortisone Sigma H6909-10ML Rapigest Waters 186001861 3x Flag Peptide Sigma F4799-4MG Anti-Flag M2 Magnetic Beads Sigma M8823-5ML Lipofectamine RNAiMAX Thermo Fisher Scientific 100014472 siFGFR3 Sigma Aldrich SIHK0780, SIHK0781, SIHK0782 native coelenterazine Biotium 10110-1 pooled siControl Dharmacon D-001810-10-20 siDaple Dharmacon L-033364-01-0005 10μM clozapine-N-oxide Cayman Chemical NC1044836 5μM native coelenterazine Biotium 10110-1 RPS6KA1 siRNA pool OriGene SR304161 non-targeting control siRNA Dharmacon D-001810-10 Triton X-100 Thermo Scientific 9002-93-1 Software and Algorithms artMS Bioconductor https://www.bioconductor.org/packages/release/bioc/html/artMS.html MSstats Bioconductor https://bioconductor.org/packages/release/bioc/html/MSstats.html Skyline MacCoss Lab https://skyline.ms/project/home/begin.view?

Techniques: Mutagenesis, Activation Assay, Binding Assay

Journal: Science (New York, N.Y.)

Article Title: A protein network map of head and neck cancer reveals PIK3CA mutant drug sensitivity

doi: 10.1126/science.abf2911

Figure Lengend Snippet: (A) Bar chart representing the ratio of helical domain (E545 and E542) mutations as compared to kinase domain mutations (H1047) across TCGA PanCancer Altas studies represented in cBioPortal (68). Line graph showing the mRNA expression (RSEM) for NRG1 across the same studies. (B) Correlation of Log2 HER3 interaction levels from AP-MS experiments and Log2 HER3 Y1197 phosphorylation levels from immunoblot analysis. All values are normalized by FLAG-PIK3CA levels in their respective experiments. Mutations marked in red were selected for in vivo experiments. (C-D) CAL-27 cells expressing inducible PIK3CA variants were transplanted into athymic nude mice. Mice were fed with doxycycline to induce PIK3CA expression. When tumor volumes reached approximately 100 mm3, mice were treated with vehicle (PBS) or CDX3379 (10mg/kg, twice a week) for approximately 15 days, as indicated. Shown are (C) tumor growth curves, (D) representative tumor images, and (C) last day tumor volume (****P < 0.0001 when compared with the control-treated group). (E) Quantification of immunoblot analysis of signaling events in the same CAL-27 cells in vitro. PIK3CA variant expression was induced by doxycycline (1μg/ml in culture medium), cells were treated with CDX3379 (1μg/ml, 1hr), and lysates were analyzed by immunoblotting as indicated. Densitometry analysis of western blots was performed using ImageJ. Data are represented as mean ± SEM, n= 3 in each group. (*P < 0.05 when compared with the control-treated group).

Article Snippet: Gαi-DREADD (pcDNA3.1) Antibodies RSK1/2/3 antibody Cell Signaling Technology 9355 ERK1/2 Cell Signaling Technology 4695 phospho-PAK1(S199/204)/PAK2(S192/197) Cell Signaling Technology 2605 PAK1 Cell Signaling Technology 2602 PAK2 Cell Signaling Technology 2608 pERK Cell Signaling Technology 9106 FGFR3 OriGene TA801078 Daple Millipore EMD ABS515 GAPDH Cell Signaling Technology 2118 secondary goat anti-rabbit HRP Southern Biotech 4010-05 P-HER3-Y1197 Cell Signaling Technology 4561 HER3 Cell Signaling Technology 12708 goat anti-mouse HRP Southern Biotech 1010-05 anti-B-tubulin Abcam ab6276 ERK Cell Signaling Technology 9102 Deposited data Unprocessed peptide files This paper PRIDE ProteomeXchange: PXD019469 Raw data This paper PRIDE ProteomeXchange: PXD019469 Chemicals, Peptides, and Recombinant Proteins Tris G-Biosciences RC108 Acetonitrile, HPLC grade (ACN) Thermo Fisher Scientific A955-4 cOmplete protease inhibitor cocktail tablets mini, EDTA-free Roche 11846 170 001 Dithiothreitol (DTT) Sigma-Aldrich 43819 Formic acid (FA) Thermo Fisher Scientific 28905 Iodoacetamide (IAA) Acros Organic 122270250 Sequencing-grade modified trypsin Promega V5111 Benzonase Sigma E1014-25KU Trifluoroacetic acid (TFA) Thermo Fisher Scientific 28904 Urea Sigma-Aldrich U5378-1kg Fetal bovine serum (FBS) Gibco A3160502 DMEM Corning MT10013CV DMEM/F12 Corning MT10092CV Water, HPLC grade Sigma-Aldrich 270733-4 L Igepal (NP-40) Sigma-Aldrich I3021 Minimal Essential Media Corning 10-009-CV Opti-MEM Thermo Fisher Scientific 31985062 BEGM™ (Lonza) Lonza CC-3170 1% Penicillin-Streptomycin Corning MT30002Cl Paraformaldehyde, 4% solution in PBS Thermo Scientific MFCD00133991 PolyJet SignaGen SL100688 Lipofectamine 3000 ThermoFisher Scientific L3000008 hydrocortisone Sigma H6909-10ML Rapigest Waters 186001861 3x Flag Peptide Sigma F4799-4MG Anti-Flag M2 Magnetic Beads Sigma M8823-5ML Lipofectamine RNAiMAX Thermo Fisher Scientific 100014472 siFGFR3 Sigma Aldrich SIHK0780, SIHK0781, SIHK0782 native coelenterazine Biotium 10110-1 pooled siControl Dharmacon D-001810-10-20 siDaple Dharmacon L-033364-01-0005 10μM clozapine-N-oxide Cayman Chemical NC1044836 5μM native coelenterazine Biotium 10110-1 RPS6KA1 siRNA pool OriGene SR304161 non-targeting control siRNA Dharmacon D-001810-10 Triton X-100 Thermo Scientific 9002-93-1 Software and Algorithms artMS Bioconductor https://www.bioconductor.org/packages/release/bioc/html/artMS.html MSstats Bioconductor https://bioconductor.org/packages/release/bioc/html/MSstats.html Skyline MacCoss Lab https://skyline.ms/project/home/begin.view?

Techniques: Expressing, Western Blot, In Vivo, In Vitro, Variant Assay

Journal: Science (New York, N.Y.)

Article Title: A protein network map of head and neck cancer reveals PIK3CA mutant drug sensitivity

doi: 10.1126/science.abf2911

Figure Lengend Snippet: Key Resources

Article Snippet: Gαi-DREADD (pcDNA3.1) Antibodies RSK1/2/3 antibody Cell Signaling Technology 9355 ERK1/2 Cell Signaling Technology 4695 phospho-PAK1(S199/204)/PAK2(S192/197) Cell Signaling Technology 2605 PAK1 Cell Signaling Technology 2602 PAK2 Cell Signaling Technology 2608 pERK Cell Signaling Technology 9106 FGFR3 OriGene TA801078 Daple Millipore EMD ABS515 GAPDH Cell Signaling Technology 2118 secondary goat anti-rabbit HRP Southern Biotech 4010-05 P-HER3-Y1197 Cell Signaling Technology 4561 HER3 Cell Signaling Technology 12708 goat anti-mouse HRP Southern Biotech 1010-05 anti-B-tubulin Abcam ab6276 ERK Cell Signaling Technology 9102 Deposited data Unprocessed peptide files This paper PRIDE ProteomeXchange: PXD019469 Raw data This paper PRIDE ProteomeXchange: PXD019469 Chemicals, Peptides, and Recombinant Proteins Tris G-Biosciences RC108 Acetonitrile, HPLC grade (ACN) Thermo Fisher Scientific A955-4 cOmplete protease inhibitor cocktail tablets mini, EDTA-free Roche 11846 170 001 Dithiothreitol (DTT) Sigma-Aldrich 43819 Formic acid (FA) Thermo Fisher Scientific 28905 Iodoacetamide (IAA) Acros Organic 122270250 Sequencing-grade modified trypsin Promega V5111 Benzonase Sigma E1014-25KU Trifluoroacetic acid (TFA) Thermo Fisher Scientific 28904 Urea Sigma-Aldrich U5378-1kg Fetal bovine serum (FBS) Gibco A3160502 DMEM Corning MT10013CV DMEM/F12 Corning MT10092CV Water, HPLC grade Sigma-Aldrich 270733-4 L Igepal (NP-40) Sigma-Aldrich I3021 Minimal Essential Media Corning 10-009-CV Opti-MEM Thermo Fisher Scientific 31985062 BEGM™ (Lonza) Lonza CC-3170 1% Penicillin-Streptomycin Corning MT30002Cl Paraformaldehyde, 4% solution in PBS Thermo Scientific MFCD00133991 PolyJet SignaGen SL100688 Lipofectamine 3000 ThermoFisher Scientific L3000008 hydrocortisone Sigma H6909-10ML Rapigest Waters 186001861 3x Flag Peptide Sigma F4799-4MG Anti-Flag M2 Magnetic Beads Sigma M8823-5ML Lipofectamine RNAiMAX Thermo Fisher Scientific 100014472 siFGFR3 Sigma Aldrich SIHK0780, SIHK0781, SIHK0782 native coelenterazine Biotium 10110-1 pooled siControl Dharmacon D-001810-10-20 siDaple Dharmacon L-033364-01-0005 10μM clozapine-N-oxide Cayman Chemical NC1044836 5μM native coelenterazine Biotium 10110-1 RPS6KA1 siRNA pool OriGene SR304161 non-targeting control siRNA Dharmacon D-001810-10 Triton X-100 Thermo Scientific 9002-93-1 Software and Algorithms artMS Bioconductor https://www.bioconductor.org/packages/release/bioc/html/artMS.html MSstats Bioconductor https://bioconductor.org/packages/release/bioc/html/MSstats.html Skyline MacCoss Lab https://skyline.ms/project/home/begin.view?

Techniques: Mutagenesis, Recombinant, Protease Inhibitor, Sequencing, Modification, Magnetic Beads, Software, Mass Spectrometry

Journal: Otology & Neurotology

Article Title: ErbB Expression, Activation, and Inhibition With Lapatinib and Tyrphostin (AG825) in Human Vestibular Schwannomas

doi: 10.1097/mao.0b013e31821f7d88

Figure Lengend Snippet: FIG. 2. Immunofluorescence staining of VS cells depicting the expression of EGFR, ErbB2, and ErbB3 receptors. A, Confocal images of individual ErbB receptors. B, Confocal images of colocalized ErbB receptors depicting predominant EGFR-ErbB2 dimerization.

Article Snippet: Cells were then blocked with 10% BSA and incubated in primary antibodies 1:100 in 4% BSA (sheep antihuman EGFR (Capralogics), mouse antihuman ErbB2 (Abcam), and rabbit antihuman ErbB3 (Santa Cruz Biotechnology) for 2 hours at room temperature in a humidified chamber.

Techniques: Immunofluorescence, Staining, Expressing

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor as a Biologic Switch in Hair Growth Cycle

doi: 10.1074/jbc.m212082200

Figure Lengend Snippet: FIG. 4. Immunoblots of crude skin samples from specific time points. a, on both Day 7 and Day 14, expression and phosphorylation of EGFR were detected in both wild type and transgenic mice. On Day 21 and Day 35, normally the telogen phase, both the expression level and phosphorylation level of EGFR were intensively down-regulated. How- ever, samples from transgenic animals had comparatively higher levels of EGFR expression and phosphorylation than samples from wild type animals. Complete down-regulation of ErbB2 and ErbB3 expression was observed on Day 35 in wild type but not in transgenic skin. Com- parable amounts of the mature form of EGF were detected at each stage in transgenic and wild type samples. b, immunoprecipitation of ErbB2 and ErbB3. On Day 14, no observable difference between wild type and transgenic skin was detected with respect to phosphorylation of ErbB2 and EGFR/ErbB2 heterodimerization. Similar results were obtained for ErbB3. On Day 35 (telogen), only transgenic skin showed EGFR/ErbB2 heterodimerization and phosphorylation.

Article Snippet: The primary antibodies and working concentrations are as follows: rabbit anti-mouse EGF (Upstate Biotechnology, Inc., 2 g/ml); mouse anti-human EGFR (Transduction Laboratories, 1 g/ml); rabbit anti-mouse ErbB2 (Santa Cruz Biotechnology, 0.2 g/ml); rabbit anti-mouse ErbB3 (Santa Cruz Biotechnology, 0.2 g/ml); and mouse anti-phosphotyrosine PY20 (Transduction Laboratories, 1 g/ml).

Techniques: Western Blot, Expressing, Phospho-proteomics, Transgenic Assay, Immunoprecipitation