Journal: Cell chemical biology

Article Title: The Advantages of Targeted Protein Degradation over Inhibition: a RTK Case Study

doi: 10.1016/j.chembiol.2017.09.009

Figure Lengend Snippet: Key Resources Table

Article Snippet: pHER3 (Y1289) , Cell Signaling , 4791.

Techniques: Ubiquitin Proteomics, Recombinant, Proliferation Assay, Software

Journal: Therapeutic Advances in Medical Oncology

Article Title: Clinicopathologic significance of nuclear HER4 and phospho-YAP(S 127 ) in human breast cancers and matching brain metastases

doi: 10.1177/1758835920946259

Figure Lengend Snippet: Biomarkers analysed by IHC.

Article Snippet: pHER3 (Y 1289 ) , Cell signaling , 21D3 , Rabbit , EDTA pH 8.8 , 1/50 , 1 h.

Techniques: Staining, Membrane

Journal: Therapeutic Advances in Medical Oncology

Article Title: Clinicopathologic significance of nuclear HER4 and phospho-YAP(S 127 ) in human breast cancers and matching brain metastases

doi: 10.1177/1758835920946259

Figure Lengend Snippet: Antibodies and conditions used for IHC analysis.

Article Snippet: pHER3 (Y 1289 ) , Cell signaling , 21D3 , Rabbit , EDTA pH 8.8 , 1/50 , 1 h.

Techniques: Incubation

Journal: Therapeutic Advances in Medical Oncology

Article Title: Clinicopathologic significance of nuclear HER4 and phospho-YAP(S 127 ) in human breast cancers and matching brain metastases

doi: 10.1177/1758835920946259

Figure Lengend Snippet: Expression and activation of HER4 in brain-metastatic breast cancers and brain metastases. (A) Chi-square analysis of cyto.HER4, nu.4ICD and m.pHER4 in major breast cancer subtypes. * p < 0.05; *** p < 0.001; **** p < 0.0001. (B) Changes in cyto.HER4, nu.4ICD and m.pHER4 in individual matched cases, separated according to major breast cancer subtypes. Chi-square p -values indicate significant differences before and after metastasis to the brain. (C) Tile plot showing overall numbers of br.MBC and BrM co-expressing pHER receptors (columns are individual tumours). (D) Representative IHC analysis of samples from a patient whose HER2+ breast cancer recurred in the brain 7 years after diagnosis and treatment with HER2-targeted therapy. This case exemplifies concomitant induction of pHER2, pHER3 and pHER4. BrM, brain metastases; br.MBC, brain metastatic breast cancers; cyto.HER4, cytoplasmic HER4; HER4, human epidermal growth factor receptor-4; IC-NST, invasive carcinoma, no special type; IHC, immunohistochemistry; LumA/B, luminal A/B; m.pHER4, membrane-pHER4; nu.4ICD, nuclear HER4-ICD.

Article Snippet: pHER3 (Y 1289 ) , Cell signaling , 21D3 , Rabbit , EDTA pH 8.8 , 1/50 , 1 h.

Techniques: Expressing, Activation Assay, Biomarker Discovery, Immunohistochemistry, Membrane

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Choice of Fixative Is Crucial to Successful Immunohistochemical Detection of Phosphoproteins in Paraffin-embedded Tumor Tissues

doi: 10.1369/jhc.2008.952911

Figure Lengend Snippet: Antiphosphoprotein antibodies

Article Snippet: The slides were washed with TBS and immersed in hematoxylin for 30 sec for counterstaining (Sigma; St. Louis, MO). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Vendor Phospho site Host species Concentration used pMET R&D (Minneapolis, MN) Y1234/Y1235 Rabbit 0.25 pg/ml pIGF-1R/IR Cell Signaling (Danvers, MA) Y1131/Y1 146 Rabbit 0.15 pg/ml pEGFR Cell Signaling Y992 Rabbit 1.0 pg/ml pHER2 Cell Signaling Y1221/Y1222 Rabbit 0.15 pg/ml pHER3 Cell Signaling Y1289 Rabbit 1.92 pg/ml pHER4 Abgent (San Diego, CA) Y1 162 Rabbit 0.62 pg/ml pAkt Cell Signaling S473 Rabbit 0.1 pg/ml pMAPK Cell Signaling T202/Y204 Rabbit 0.5 ng/ml pTyr R&D pan T Mouse 2 pg/ml pp53 Abcam (Cambridge, MA) S315 Rabbit 0.2 pg/ml Open in a separate window Antiphosphoprotein antibodies To confirm the specificity of phosphoprotein staining, lambda phosphatase was applied on the sections before incubation with primary antibodies.

Techniques: Concentration Assay

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Choice of Fixative Is Crucial to Successful Immunohistochemical Detection of Phosphoproteins in Paraffin-embedded Tumor Tissues

doi: 10.1369/jhc.2008.952911

Figure Lengend Snippet: IHC staining of phosphoproteins in xenograft tumor tissues

Article Snippet: The slides were washed with TBS and immersed in hematoxylin for 30 sec for counterstaining (Sigma; St. Louis, MO). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Vendor Phospho site Host species Concentration used pMET R&D (Minneapolis, MN) Y1234/Y1235 Rabbit 0.25 pg/ml pIGF-1R/IR Cell Signaling (Danvers, MA) Y1131/Y1 146 Rabbit 0.15 pg/ml pEGFR Cell Signaling Y992 Rabbit 1.0 pg/ml pHER2 Cell Signaling Y1221/Y1222 Rabbit 0.15 pg/ml pHER3 Cell Signaling Y1289 Rabbit 1.92 pg/ml pHER4 Abgent (San Diego, CA) Y1 162 Rabbit 0.62 pg/ml pAkt Cell Signaling S473 Rabbit 0.1 pg/ml pMAPK Cell Signaling T202/Y204 Rabbit 0.5 ng/ml pTyr R&D pan T Mouse 2 pg/ml pp53 Abcam (Cambridge, MA) S315 Rabbit 0.2 pg/ml Open in a separate window Antiphosphoprotein antibodies To confirm the specificity of phosphoprotein staining, lambda phosphatase was applied on the sections before incubation with primary antibodies.

Techniques: Immunohistochemistry

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Choice of Fixative Is Crucial to Successful Immunohistochemical Detection of Phosphoproteins in Paraffin-embedded Tumor Tissues

doi: 10.1369/jhc.2008.952911

Figure Lengend Snippet: IHC staining of phosphoproteins in xenograft and human clinical tumor tissues. (A) SKOV-3 xenograft tumor tissues fixed in Streck’s tissue fixative (STF), 10% formalin, and 4% paraformaldehyde (PFA) were stained with anti-pMet antibody. (B) Human lung tumor tissue fixed in 10% formalin and STF were stained with anti-pHER4 antibody. Bar = 50 μm.

Article Snippet: The slides were washed with TBS and immersed in hematoxylin for 30 sec for counterstaining (Sigma; St. Louis, MO). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Vendor Phospho site Host species Concentration used pMET R&D (Minneapolis, MN) Y1234/Y1235 Rabbit 0.25 pg/ml pIGF-1R/IR Cell Signaling (Danvers, MA) Y1131/Y1 146 Rabbit 0.15 pg/ml pEGFR Cell Signaling Y992 Rabbit 1.0 pg/ml pHER2 Cell Signaling Y1221/Y1222 Rabbit 0.15 pg/ml pHER3 Cell Signaling Y1289 Rabbit 1.92 pg/ml pHER4 Abgent (San Diego, CA) Y1 162 Rabbit 0.62 pg/ml pAkt Cell Signaling S473 Rabbit 0.1 pg/ml pMAPK Cell Signaling T202/Y204 Rabbit 0.5 ng/ml pTyr R&D pan T Mouse 2 pg/ml pp53 Abcam (Cambridge, MA) S315 Rabbit 0.2 pg/ml Open in a separate window Antiphosphoprotein antibodies To confirm the specificity of phosphoprotein staining, lambda phosphatase was applied on the sections before incubation with primary antibodies.

Techniques: Immunohistochemistry, Staining

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Choice of Fixative Is Crucial to Successful Immunohistochemical Detection of Phosphoproteins in Paraffin-embedded Tumor Tissues

doi: 10.1369/jhc.2008.952911

Figure Lengend Snippet: IHC staining of phosphoproteins in human clinical tumor tissues

Article Snippet: The slides were washed with TBS and immersed in hematoxylin for 30 sec for counterstaining (Sigma; St. Louis, MO). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Vendor Phospho site Host species Concentration used pMET R&D (Minneapolis, MN) Y1234/Y1235 Rabbit 0.25 pg/ml pIGF-1R/IR Cell Signaling (Danvers, MA) Y1131/Y1 146 Rabbit 0.15 pg/ml pEGFR Cell Signaling Y992 Rabbit 1.0 pg/ml pHER2 Cell Signaling Y1221/Y1222 Rabbit 0.15 pg/ml pHER3 Cell Signaling Y1289 Rabbit 1.92 pg/ml pHER4 Abgent (San Diego, CA) Y1 162 Rabbit 0.62 pg/ml pAkt Cell Signaling S473 Rabbit 0.1 pg/ml pMAPK Cell Signaling T202/Y204 Rabbit 0.5 ng/ml pTyr R&D pan T Mouse 2 pg/ml pp53 Abcam (Cambridge, MA) S315 Rabbit 0.2 pg/ml Open in a separate window Antiphosphoprotein antibodies To confirm the specificity of phosphoprotein staining, lambda phosphatase was applied on the sections before incubation with primary antibodies.

Techniques: Immunohistochemistry

Journal: Cell chemical biology

Article Title: The Advantages of Targeted Protein Degradation over Inhibition: a RTK Case Study

doi: 10.1016/j.chembiol.2017.09.009

Figure Lengend Snippet: Key Resources Table

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies EGFR SantaCruz 1005 FLAG Sigma F1804 Tubulin Sigma T9026 HER2 Cell Signaling 2165S p-EGFR (Y1068) Abcam ab40815 p-HER2 (Y1221/1222) Cell Signaling 2243S p-AKT (T308) Cell Signaling 2965S p-ERK 1/2 (T202/204) Cell Signaling 4370 HER3 Cell Signaling 12708 pHER3 (Y1197) Cell Signaling 4561 pHER3 (Y1289) Cell Signaling 4791 pGSK-3b (S9) Cell Signaling 9331 c-Met Cell Signaling 8198 c-Met Cell Signaling 3127 p-Met (Y1245/1235) Cell Signaling 3126 p-AKT (S473) Cell Signaling 4060 Ubiquitin (P4D1) Cell Signaling 3936 p230 Trans Golgi BD Biosciences 611281 EEA1 BD Biosciences 610456 Clathrin Heavy Chain SantaCruz sc-12734 Alexa Fluor-546 conjugated anti-mouse ThermoFisher A-21143 Alexa Fluor-488 conjugated anti-rabbit ThermoFisher A-11008 HRP linked Mouse IgG GE Life Sciences NA931 HRP Linked Rabbit IgG GE Life Sciences NA934 Chemicals, Peptides, and Recombinant Proteins Cycloheximide Sigma C104450 MLN4924 Sigma 5.05477 PR-619 LifeSensors SI9619 EZ-link Sulfo-NHS-SS-Biotin Thermo 21331 Pierce NeutrAvidin Agarose beads Thermo 29200 Agarose-TUBE 1 LifeSensors UM401 Protein A-Sepharose 4B, Fast Flow beads Sigma P9424 Recombinant Human HGF Protein R&D Systems 294-HG-250 Critical Commercial Assays CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) Promega G5421 Experimental Models: Cell Lines OVCAR8 Joyce Liu, Dana Farber MDA-MB-231 ATCC HTB-26 HeLa ATCC CCL-2 HCC827 ATCC CRL-2868 H3255 Katerina Politi, Yale H1975 ATCC CRL-5908 SKBr3 ATCC HTB-30 GTL16 F. Maina, Developmental Biology Institute of Marseille-Luminy Hs746T ATCC HTB-135 Oligonucleotides Clathrin Heavy Chain siRNA SantaCruz sc-35067 Software and Algorithms Image Lab 6.0 Biorad N/A Graphpad Prism N/A Open in a separate window Key Resources Table Contact for Reagent and Resource Sharing Further information and request for resources and reagents should be directed and will be fulfilled by Craig Crews ( ude.elay@swerc.giarc ).

Techniques: Ubiquitin Proteomics, Recombinant, Proliferation Assay, Software