Anti erbb2

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-ErbB2 is a lab equipment product produced by Cell Signaling Technology. It is an antibody that specifically targets the ErbB2 protein, also known as HER2. The core function of this product is to detect and quantify the ErbB2 protein in various research applications.

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16 protocols using anti erbb2

Sema3e and Sema3d Protein Interactions

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Human recombinant Sema3e (#3239-S3-025), and human recombinant Sema3d were obtained from R&D Systems. Anti-phospho-Akt (#4060), anti-Akt (#9272), anti-β-actin (#4967), anti-ErbB2 (#4290), anti-ErbB2 (#2165), and anti-phospho-ErbB2 (#2247) were purchased from Cell Signaling, along with anti-Nrp (Santa Cruz, #7239), anti-Nrp (Abcam, #81321), anti-V5 (Life technologies, #R960), anti-ENOS (BD Biosciences, #610296) and anti-GFP (Abcam, #AB6673). Lipofectamine 2000 (Invitrogen) was used as the transfection reagent for HEK293T and Cos-7 cells. Lipofectamine RNAiMAX (Invitrogen, 13778030) was used for siRNA transfections.

Aghajanian H., Cho Y.K., Manderfield L.J., Herling M.R., Gupta M., Ho V.C., Li L., Degenhardt K., Aharonov A., Tzahor E, & Epstein J.A. (2016). Coronary vasculature patterning requires a novel endothelial ErbB2 holoreceptor. Nature Communications, 7, 12038.

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Fluorescence Imaging of Receptor Localization

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Cells were cultured to ~70% confluence on glass coverslips, washed with PBS, and incubated with 2% BSA in PBS for 30 min to block non-specific binding. 1 μM of Cy5.5-labeled peptides were added and incubated for 30 min at 4°C. The cells were then washed 3X with PBS, fixed with ice cold 4% paraformaldehyde (PFA) for 10 min, washed with PBS 1X, and then mounted on glass slides with ProLong Gold reagent containing DAPI (Invitrogen). For antibody staining, cells were incubated with either anti-EGFR (1:500, #2232S) or anti-ErbB2 (1:500, #29D8) primary antibody from Cell Signaling Inc overnight at 4°C after fixation, and then washed with PBS 3X and processed with secondary antibody staining. Either goat anti-rabbit IgG (H+L) labeled with Alexa-Fluor 488 (1:1000, #A-11008, Invitrogen) or goat anti-mouse IgG (H&L) labeled with Alexa-Fluor 568 (1:1000, #ab175473, Abcam) was added and incubated for 1 hour at room temperature (RT). The cells were washed with PBS 3X, and mounted onto glass coverslips. Confocal fluorescence images were collected using DAPI, AF488, AF568 and Cy5.5 filter sets. Fluorescence intensities from 3 independent images were quantified using custom Matlab (Mathworks) software.

Chen J., Zhou J., Gao Z., Li X., Wang F., Duan X., Li G., Joshi B.P., Kuick R., Appelman H.D, & Wang T.D. (2018). Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo. Journal of medicinal chemistry, 61(12), 5323-5331.

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Immunofluorescence Staining of Muscle Tissue

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Transverse muscle sections were fixed with 4% PFA for 5 min at room temperature and washed twice with PBS. The permeabilization was performed in methanol for 3 min at −20 °C. Slices were then washed twice in a solution of PBS and 0.1 M glycine for 5 min and incubated for 40 min at room temperature in a solution of PBS, 2% goat serum (DAKO) and 1% BSA. The primary antibodies were incubated overnight in PBS with 1% BSA: anti-ErbB2 (1:200, #2165, Cell Signaling); anti-ErbB3 (1:200, #12708, Cell Signaling), anti-dystrophin (1:400, #D8168, Sigma). For Pax 7 (1:10, purchased by Hybridoma Bank) staining an additional step was performed after the permeabilization: slides were incubated 7 minutes at 500 W in microwave in a hot antigen retrieval solution (10 mM citrate buffer pH 6, obtained from 0.1 M citric acid and 0.1 M sodium citrate) and then washed three times with PBS. The secondary antibodies used were goat-anti-mouse Alexa Fluor 488 (1:200, Invitrogen) and goat-anti-rabbit Cy3 (1:400, Jackson). Nuclei were stained with DAPI (1:1000, Sigma).

Morano M., Ronchi G., Nicolò V., Fornasari B.E., Crosio A., Perroteau I., Geuna S., Gambarotta G, & Raimondo S. (2018). Modulation of the Neuregulin 1/ErbB system after skeletal muscle denervation and reinnervation. Scientific Reports, 8, 5047.

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Western Blot Analysis of Receptor Signaling

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Cells were lysed with RIPA lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate) supplemented by protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails (Calbiochem). Lysates were separated on 7.5% or 8% Tris-Glycine SDS-polyacrylamide gel and were transferred to PVDF membranes (Millipore). The membranes were blocked with 5% skim milk (Bio-Rad) dissolved in TBST buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20). Then, the membranes were incubated with primary antibodies overnight at 4°C. Anti-EGFR antibody (#A300-388A) was purchased from Bethyl Laboratories. Anti-β-actin antibody (#A5441) and anti-γ-tubulin antibody (#A9044) were purchased from Sigma-Aldrich. All other antibodies including anti-phospho EGFR Y1068 (#3777), anti-phospho ERBB2 Y1221/1222 (#2243), anti-ERBB2 (#2165), anti-phospho SRC Y416 (#6943), anti-SRC (#2109), anti-phospho-ERK 1/2 T202/Y204 (#4370), anti-ERK 1/2 (#4695), anti-phospho AKT S473 (#4060), and anti-AKT (#9272) were purchased from Cell Signaling Technologies. Horseradish peroxidase-conjugated secondary antibodies (anti-rabbit: #31460, anti-mouse: #31430, Pierce) and SuperSignal West Pico Chemiluminescent Substrate (Pierce) were used to detect signals.

Hong Y.S., Kim J., Pectasides E., Fox C., Hong S.W., Ma Q., Wong G.S., Peng S., Stachler M.D., Thorner A.R., Van Hummelen P, & Bass A.J. (2014). Src Mutation Induces Acquired Lapatinib Resistance in ERBB2-Amplified Human Gastroesophageal Adenocarcinoma Models. PLoS ONE, 9(10), e109440.

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Chromatin Immunoprecipitation Protocol

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Chromatin immunoprecipitation assays were performed using a Pierce™ Magnetic ChIP Kit (Thermo Scientific™). Immunoprecipitation was performed with anti-ER-α, anti-ERBB2, and anti-PR antibodies (Cell Signaling Technology). Specific regions were quantified via qRT-PCR using the primers listed in Table S13.

Zhao Z., Li L., Du P., Ma L., Zhang W., Zheng L., Lan B., Zhang B., Ma F., Xu B., Zhan Q, & Song Y. (2019). Transcriptional Downregulation of miR-4306 serves as a New Therapeutic Target for Triple Negative Breast Cancer. Theranostics, 9(5), 1401-1416.

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Western Blot Analysis of Signaling Proteins

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This assay was performed as previously described [54 (link)]. The following antibodies were used: anti-ErbB2 (Cell Signaling Technology), anti-Erk (Cell Signaling Technology), anti-phospho-Erk (Cell Signaling Technology), anti-Mek (Cell Signaling Technology), anti-Atg12 (Cell Signaling Technology), anti-β-actin (Santa Cruz Biotechnology), anti-GAPDH (Cell Signaling Technology), anti-CDK4 (Santa Cruz Biotechnology).

Khan I.A., Yoo B.H., Rak J, & Rosen K.V. (2017). Mek activity is required for ErbB2 expression in breast cancer cells detached from the extracellular matrix. Oncotarget, 8(62), 105383-105396.

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Immunoprecipitation and Western Blot Analysis of MUC4 and ErbB2

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T3M4 WT and SC cells were lysed in non-denaturing RIPA buffer containing protease inhibitor. 500μg of proteins were incubated with either mouse anti-MUC4 (3μg/ml, 8G7) or Mouse IgG (3μg/ml; Jackson ImmunoResearch Laboratories, Inc, USA) overnight at 4°C. Lysates were then incubated with protein G Sepharose beads (GenScript, NJ, USA) for 2 h at room temperature. The beads were washed three times with RIPA buffer and boiled in SDS loading buffer (1X). Equal amounts of samples were subjected to 4–20% gradient (Bio-Rad, CA, USA) SDS-PAGE gel electrophoresis and transferred to PVDF membrane. The membranes were blocked with 5% skimmed milk and incubated with primary antibody rabbit anti-ErbB2 (Cell Signaling Technology, USA, 1:1000) and anti-Mouse IgG (Jackson ImmunoResearch Laboratories, Inc, PA, USA, 1:4000) overnight. After incubating with respective secondary antibodies, the protein-antibody complex was detected using enhanced chemiluminescence (Bio-Rad, USA).

Sagar S., Leiphrakpam P.D., Thomas D., McAndrews K.L., Caffrey T.C., Swanson B.J., Clausen H., Wandall H.H., Hollingsworth M.A, & Radhakrishnan P. (2021). MUC4 enhances gemcitabine resistance and malignant behaviour in pancreatic cancer cells expressing cancer-associated short O-glycans. Cancer letters, 503, 91-102.

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Recombinant PEPD Protein Expression and Characterization

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hPEPD, hPEPD-G278D, hPEPD-R265X and hPEPD-X265R were produced in E.coli as previously described [13 (link), 17 (link)]. EP and human EGF (236-EG-200) were purchased from Fresenius Kabi and R&D Systems, respectively. The following antibodies were used in the study: anti-PEPD (Abcam, ab86507) which detects hPEPD, hPEPD-G278D and mPEPD, anti-ErbB1 (Cell Signaling, 2232), anti-p-ErbB1 (Y1173) (Cell Signaling, 4407), anti-ErbB2 (Cell Signaling, 2165), anti-p-ErbB2 (Y1221/1222) (Cell Signaling, 2243), anti-AKT (Cell Signaling, 4691), anti-p-AKT (Cell Signaling, 4060), anti-ERK (Cell Signaling, 9102), anti-p-ERK (Cell Signaling, 9101), anti-STAT3 (Cell Signaling, 4904), anti-p-STAT3 (Cell Signaling, 9145), anti-cleaved caspase-3 (Cell Signaling, 9661), anti-BCL-2 (Cell Signaling, 2870), anti-BAX (Cell Signaling, 2772), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Millipore, MAB374), and biotin-conjugated anti-6XHistidines (His)-tag (Bethyl, A190-113B). Horseradish peroxidase (HRP)-conjugated streptavidin (N100) was purchased from Thermo Scientific. A goat anti-rabbit IgG-HRP was purchased from Jackson ImmunoResearch (111-035-003).

Yang L., Li Y., Bhattacharya A, & Zhang Y. (2016). Dual inhibition of ErbB1 and ErbB2 in cancer by recombinant human prolidase mutant hPEPD-G278D. Oncotarget, 7(27), 42340-42352.

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Quantitative RT-PCR and Protein Analysis

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RNA was extracted from cultured cells and tumors using RNeasy Purification Kit (QIAGEN). Quantitative real-time RT-PCR amplification was with Taqman one-step RT-PCR reagents and results were normalized to co-amplified GAPDH. Primers and probes are listed as below: PSA: Forward, 5′-GATGAAACAGGCTGTGCCG-3′; Reverse, 5′-CCTCACAGCTACCCACTGCA-3′; Probe, 5′-FAM-CAGGAACA AAAGCGTGATCTTGCTGGG-3′. TMPRSS2: Forward, 5′-CCTGTGTGCCAAGACGACTG-3′; Reverse, 5′-TTATAGCCCATGTCCCTGCAG-3′; Probe, 5′-FAM-AACGAGAACTACGGGCGGGCG-3′. AR: Forward, 5′-GGAATTCCTGTGCATGAAA-3′; Reverse, 5′-CGAAGTTCATCAAAGAATT-3′; Probe, 5′-FAM-CTTCAGCATTATTCCAGTG-3′. FKBP5 primers were directly purchased from Thermal Fisher Scientific. Proteins were extracted by boiling for 15 min in 2% SDS and detected by blotting with anti-AR, anti-β-tubulin (Millipore), anti-PSA (BioDesign), anti-ErbB3(p-Tyr1289), anti-AKT(p-Ser473), anti-ErbB2, anti-ErbB3, anti-p110α, anti-p110β (Cell Signaling), or anti-β-actin (Abcam).

Gao S., Ye H., Gerrin S., Wang H., Sharma A., Chen S., Patnaik A., Sowalsky A.G., Voznesensky O., Han W., Yu Z., Mostaghel E.A., Nelson P.S., Taplin M.E., Balk S.P, & Cai C. (2016). ErbB2 Signaling Increases Androgen Receptor Expression in Abiraterone-Resistant Prostate Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(14), 3672-3682.

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Antibody-Based Protein Analysis Protocol

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This assay was performed as described previously [10 (link)]. The following antibodies were used in this study: Anti-Irf6, anti-Blnk, anti-ErbB2, anti-RSK, and anti-phospho-RSK (all from Cell Signaling Technology, Danvers, MA, USA), anti-ΔNp63 and anti-TAp63 (BioLegend, San Diego, CA, USA), anti-CDK4 (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-β-actin (Santa Cruz Biotechnology and Sigma-Aldrich).

Khan I.A., Yoo B.H., McPhee M., Masson O., Surette A., Dakin-Hache K., Younis T., Bethune G, & Rosen K.V. (2018). ErbB2-driven downregulation of the transcription factor Irf6 in breast epithelial cells is required for their 3D growth. Breast Cancer Research : BCR, 20, 151.

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