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2 protocols using duolink 2 assay kit

1

Multimodal Analysis of EGFR and HER2 Signaling

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Anti-EGFR, anti-phospho-EGFR, anti-phospho-HER2, anti-HER2, anti-phopho-akt, anti-akt monoclonal antibodies were purchased from Cell Signaling Technology. Anti-GAPDH monoclonal antibody (sc-365052) was purchased from Santacruz Biotechnology (Dallas, TX, USA). Cell-titer Glo assay kit was purchased from Promega (Madision, MI, USA). HER2 protein and EGFR protein for surface plasmon resonance study was obtained from Abcam and Leinco Technologies Inc, respectively. 1× RBC lysis buffer (420301, Biolegend, San Diego, CA); Concanavalin A (ConA; C5275, Sigma-Aldrich, Saint Louis, MO, USA); viability dye (LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, L34975, Invitrogen, Eugene, OR, USA); Fixation/Permeabilization solution (00-5123-43 and 00-5223-56, eBioscience, Carlsbad, CA, USA); CD3-PerCP-eFlour710 (17A2, Invitrogen); CD4-FITC (GK 1.5, BD Biosciences); CD8a-BV650 (53–6.7, BD Horizon); and Ki67-AF647(B56, BD Pharmingen) for peptide immunogenicity study were purchased. Proximity ligation assay was performed using the Duolink® II assay kit (Sigma Aldrich). Primary antibodies for EGFR, HER2 and HER3 were from Enzo Life Sciences.
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2

Doxorubicin-Loaded Nano-Formulation for HER2+ Cancer

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Cancer cell lines BT-474, A549, Calu-3, and MCF-7 and media RPMI1640 were purchased from ATCC (Manassas, VA, USA). BT-474 is a HER2 positive breast cancer cell line; Calu-3 and A549 are HER2 positive lung cancer cell lines. MCF-7 is a HER2 negative breast cancer cell line. Non-cancerous human lung fibroblast cells (HLFs) and fibroblast basal medium were also purchased from ATCC (Manassas, VA, USA). Doxorubicin was purchased from Astatech. Lipid Dipalmitoylphosphatidylcholine (DPPC) (Avanti Polar Lipids Inc., Birmingham, AL, USA), Cholesterol (Millipore Sigma, Burlington, MA, USA), Poly(ethylene glycol) distearoylphosphatidylethanolamine (PEG-DSPE) (Lysan Bio. Inc., Arab, AL, USA) were purchased. Sephadex G-25 was from Millipore Sigma, CellTiter-Glo was from Promega, Amicron Ultra was from EMD Millipore, Spectrum™ Spectra/Por™ 1 RC dialysis membrane tubing (6000 to 8000 Dalton Molecular Weight cutoff (MWCO)) was from Fisher Scientific (Waltham, MA, USA). Athymic nude mice (Foxn1-nude, female, 6–7 weeks old) were purchased from ENVIGO Laboratories (Indianapolis, IN, USA). Luciferase transfected A549 cells (A549 Red-FLuc NSCLC cells) were from Perkin Elmer. Proximity ligation assay was performed using the Duolink® II assay kit (Sigma Aldrich, St. Louis, MO, USA). Primary antibodies for EGFR, HER2, and HER3 were from Enzo Life Sciences (Farmingdale, NY, USA).
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