293T, E0771, and JAWSII cells were grown on 8-well slides (MatTek) coated with 1 µg/mL human fibronectin (EMD Millipore). Transfection was performed using Lipofectamine3000 (Thermofisher). For Lysotracker (ThermoFisher) staining, cells were incubated with 75 nm Lysotracker for 1 hour at 37°C. For endoplasmic reticulum (ER) staining, cells were incubated with 2 μL ER CellLight ER-RFP Bacman V.2.0 (Thermofisher) for 24 hours. Slides were washed with phosphate buffered saline (PBS), fixed with 10% formalin for 20 min and mounted with DAPI Fluoromount-G (SouthernBiotech). Staining was performed in 0.1% PBST with rabbit anti-HER2 (1:200, Cell Signaling), rat anti-LAMP1 (1:200, Abcam) or rat anti-MHCII (1:200, Thermofisher) at 4° overnight and antirabbit Alexa Fluor 488 (1:1000 dilution, Invitrogen) or antirat Texas Red-X (1:1000, invitrogen) at room temperature for 1 hour. Slides were mounted with Fluoromount-G with DAPI (Southern Biotech).
Rabbit anti her2
Manufactured by Cell Signaling Technology
Sourced in United Kingdom, United States
Rabbit anti-HER2 is a primary antibody that specifically recognizes the human epidermal growth factor receptor 2 (HER2) protein. It is designed for use in various immunoassay applications to detect and study the expression of HER2.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti akt, by Cell Signaling Technology (2 mentions)
Rabbit anti her3, by Cell Signaling Technology (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Lysotracker, by Thermo Fisher Scientific (1 mentions)
Human fibronectin, by Merck Group (1 mentions)
Texas red x, by Thermo Fisher Scientific (1 mentions)
Rat anti mhcii, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Rat anti lamp1, by Abcam (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Anti mouse irdye 680, by LI COR (1 mentions)
Semi dry transfer system, by Bio-Rad (1 mentions)
Anti rabbit irdye 800, by LI COR (1 mentions)
Mini protean system, by Bio-Rad (1 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (1 mentions)
Rabbit anti cyclin d1, by Cell Signaling Technology (1 mentions)
Rabbit anti phospho akt ser473, by Merck Group (1 mentions)
Recombinant human egf, by R&D Systems (1 mentions)
Bkm120, by Active Motif (1 mentions)
6 protocols using rabbit anti her2
1
Immunofluorescence Imaging of HER2, LAMP1, and MHC-II
2
Western Blot Analysis of HER2 and OVA
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Tissue culture cells were harvested and lysed using RIPA buffer. Total lysate (20 µg) was ran on a gel using the Biorad Mini-PROTEAN system, followed by transfer using the Biorad semi dry transfer system. Primary antibodies used were rabbit anti-HER2 (1:1000, Cell Signaling), mouse anti-OVA (1:1000, Abcam) or rabbit anti-GAPDH (1:1000, Cell Signaling). Antirabbit IRDye 800 (1:1000, LICOR) and antimouse IRDye 680 (1:1000, LICOR) were used as secondaries. Blocking, probing, visualization and quantification were performed according to LI-COR Odyssey system specifications.
3
Immunoblotting Antibodies and Reagents
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Antibodies for Western blot were as follows: mouse anti-EGFR and mouse anti-HER4 (Abcam, Cambridge, UK), rabbit anti-HER3, mouse anti-p85 PI3K, rabbit anti-phospho EGFR (Try1068), rabbit anti-HER2, rabbit anti-cyclinD1, mouse anti-CDK4, rabbit anti-phospho p44/42 MAPK (p- Erk1/2), rabbit anti-p44/42 MAPK (Erk1/2), rabbit anti-Akt (Cell signaling technology, Danvers, MA), rabbit anti-phospho HER2 and rabbit anti-phospho Akt (Ser473) (Sigma-Aldrich, MO, USA). Recombinant human EGF was purchased from R&D systems, MN, USA. Varlitinib was supplied by ASLAN Pharmaceuticals, Singapore. BKM-120 was purchased from Active Biochem, NJ, USA. These were dissolved as stock in 100% dimethyl sulfoxide (DMSO) and stored at −80°C until use.
4
HER2 and pJNK Colocalization in SNU-216 Cells
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SNU-216 cells (1 × 104 cells/well) were cultured on 4-well chamber slide (Thermo Scientific, Rockford, IL, United States). After 24 h, cells were fixed with 4% paraformaldehyde for 10 min, and blocked with 5% normal donkey serum containing 0.5% Triton X-100 for 5 min. Cells were incubated overnight at 4 °C with mixture of the following primary antibodies: rabbit anti-HER2 (1:200; Cell Signaling Technology) and mouse anti-pJNK (1:200, Santa Cruz Biotechnology). Alexa fluor-555-conjugated anti-rabbit IgG (1:200, Life Technologies) and Alexa fluo-488-conjugated anti-mouse IgG (1:200, Life Technologies) were used as secondary antibodies. To examine whether JNK inhibition reorganizes cytoskeleton, filamentous actin (F-actin) was visualized. Cells were incubated with 165 nmol/L Alexa Fluor-633-conjugated phalloidin (Invitrogen, Carlsbad, CA, United States) for 10 min, followed by 4’6’-diamidio-2-phenoylindole (DAPI) staining. Immunofluorescence was observed under a fluorescence microscope.
5
Characterization of Cell Line Responses
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Cell lines. SKOV3, OVACAR5, MDA-MB-231 and MDA-MB-361 cells [from the American Tissue Culture Collection (ATCC) Manassas, VA, USA] were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, and 1% streptomycin/penicillin at 37̊C in a humidified incubator containing 5% CO 2 in air.
Antibodies and chemical reagents. Mouse anti-β-actin antibody, dimethyl sulfoxide (DMSO), glycerol, glycine, sodium chloride, Trizma base and Tween-20 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-PARP1, rabbit anti-FOXO3, mouse anti-LaminA/C antibodies and auranofin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-Her3, rabbit anti-Her2, rabbit anti-phospho-FOXO3, rabbit anti-phospho-Akt, rabbit anti-Akt, rabbit anti-caspase-3, rabbit anti-Bax, rabbit anti-Bim and rabbit anti-Bcl2 antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). The rabbit anti-MUC4 antibody was purchased from Abcam (Cambridge, MA). Goat anti-mouse and goat anti-rabbit horseradish peroxidase-conjugated IgG were obtained from Jackson ImmunoResearch (West Grove, PA, USA). ECL Western Blotting Detection Reagents were obtained from GenDEPOT (Barker, TX, USA).
Antibodies and chemical reagents. Mouse anti-β-actin antibody, dimethyl sulfoxide (DMSO), glycerol, glycine, sodium chloride, Trizma base and Tween-20 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-PARP1, rabbit anti-FOXO3, mouse anti-LaminA/C antibodies and auranofin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-Her3, rabbit anti-Her2, rabbit anti-phospho-FOXO3, rabbit anti-phospho-Akt, rabbit anti-Akt, rabbit anti-caspase-3, rabbit anti-Bax, rabbit anti-Bim and rabbit anti-Bcl2 antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). The rabbit anti-MUC4 antibody was purchased from Abcam (Cambridge, MA). Goat anti-mouse and goat anti-rabbit horseradish peroxidase-conjugated IgG were obtained from Jackson ImmunoResearch (West Grove, PA, USA). ECL Western Blotting Detection Reagents were obtained from GenDEPOT (Barker, TX, USA).
6
Protein Expression Analysis by Western Blot
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Effects of antibody treatments on total and phospho-protein levels were investigated by Western blot analysis. Primary antibodies: Rabbbit anti-EGFR, rabbit anti-HER2, rabbit anti-HER3 (Cell Signaling Technology, clone #: D38B1, 29D8, and D22C5), mouse anti-HER3 (Millipore, clone #: 2F12), rabbit anti-AKT, rabbit anti-pAKT (Ser473), rabbit anti-ERK1/2, rabbit anti-pERK1/2 (Thr202/204), and rabbit anti-βactin (Cell Signaling Technology, clone #: D9E, D13.14.4E, and 13E5) diluted in Odyssey Blocking Buffer (LI-COR Biosciences) were used for detection, and membranes were scanned on Odyssey Scanner (LI-COR Biosciences) using Odyssey IR Imaging System (LI-COR Biosciences).
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