Protein was extracted with RIPA buffer with complete protease inhibitors (Roche), separated by electrophoresis, transferred to PVDF Membrane (Millipore), and blocked with 5% skim milk (BD). The primary antibodies, EGFR (1005) (Santa Cruz), p-EGFR (Tyr 1173) (Santa Cruz), Sox2 (R&D systems), Nestin (BD), GFAP (ImmunO), NICD (Cell signaling), Hes1 (Millipore), Hey1 (abcam), PEDF (Upstate), and β-actin (Santa Cruz) were incubated overnight at 4°C. Immunoreactive bands were visualized using peroxidase-labeled affinity purified secondary antibodies (KPL) and the detection reagent Amersham ECL prime western blotting detection reagent (GE Healthcare).
Egfr 1005
Manufactured by Santa Cruz Biotechnology
Sourced in United Kingdom, United States
EGFR (1005) is a laboratory product offered by Santa Cruz Biotechnology. It is a recombinant protein that corresponds to the extracellular domain of the human epidermal growth factor receptor (EGFR). This protein is commonly used in research applications involving EGFR-related signaling pathways and receptor-ligand interactions.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
E cadherin, by Santa Cruz Biotechnology (2 mentions)
Erbb3 c 17, by Santa Cruz Biotechnology (2 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
P egfr tyr 1173, by Santa Cruz Biotechnology (1 mentions)
Nestin, by BD (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Amersham ecl prime, by Cytiva (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Gapdh, by Merck Group (1 mentions)
Traf6, by Merck Group (1 mentions)
P27kip1 c 19, by Santa Cruz Biotechnology (1 mentions)
Vegfa a 20, by Santa Cruz Biotechnology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Srx 101c, by Konica Minolta (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
9 protocols using egfr 1005
1
Protein Expression Analysis in Stem Cells
2
Western Blot Analysis of Protein Targets
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Cells harvested at different time points were lysed and total protein extracts were used for western blotting using antibodies specific for AR (US Biological, Salem, MA), PSA (Santa Cruz Biotechnology, Dallas, TX), Cbl (C-15) (Santa Cruz Biotechnology, Dallas, TX), TRAF6 (Millipore, Temecula, CA), p27Kip1 (C-19) (Santa Cruz, Biotechnology), IRAK1 (F-4) (Santa Cruz, Biotechnology), ZFAND1 (A-14) (Santa Cruz Biotechnology), FGD4 (Epitomics, Burlingame, CA), ABHD3 (Biorbyt, Cambrige, UK), DOK4 (C-16) (Santa Cruz Biotechnology), EGFR (1005) (Santa Cruz Biotechnology), VEGFA (A-20) (Santa Cruz Biotechnology), α-tubulin (Cell Signaling, Danvers, MA), GAPDH (Sigma-Aldrich, St. Louis, MO). Total extracts (30–50 μg) were directly mixed with Lammeli sample buffer and separated on SDS-PAGE. Immunoblotting was performed using appropriate primary and horseradish peroxidase conjugated respective secondary antibodies. Positive signals were detected using a chemiluminiscence ECL kit (Pierce, Rockford, IL).
3
Western Blot Analysis of Cell Signaling
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Cultured cells were lysed in ice-cold lysis buffer containing 20mM TRIS pH7.5, 150mM NaCl, 1% NP40, 5mM EDTA pH 8.0, 1% NaF, 1% NaVO4, 0.1% NH4 Molybate and 8% Complete phosphatase and protease inhibitor (Roche). The lysates were centrifuged and supernatant was collected for Western blot analysis and stored at −80°C. Protein lysate was separated by SDS-PAGE and transferred to PVDF membrane (Millipore). The membrane was blocked in 5% milk in PBS/0.1% Tween solution for Llgl1 antibody, 5% BSA in 10x TBS solution for Integrin α6 antibody, or 3% BSA in TBS/0.1% Tween solution for all other antibodies and then used for immunoblotting. Proteins on the membrane were treated with SuperSignal West Pico Chemiluminescent Substrate (Pierce), visualized on Blue Autoradiography film (GeneMate) and developed with a Konica SRX-101C. Antibodies included Llgl1 (Abnova H00003996-M01), E-cadherin (H-108; Santa Cruz sc-7870), dpERK 1&2 (Sigma M8159), ERK 1&2 (Cell Signaling Technologies 4370), pStat3 (Tyr705; Cell Signaling Technologies 9145), Stat3 (124H6; Cell Signaling Technologies 9139), pAkt (Ser473; Cell Signaling 4060), Akt (Cell Signaling 9272), TAZ (V386; Cell Signaling 4883), Slug (C19G7; Cell Signaling 9585), EGFR 1005 (Santa Cruz sc-03), and βactin (Sigma Aldrich A5441). Integrin α6, was a kind gift from Dr. Anne Cress.
4
Antibody Immunoblotting Protocol
5
Immunofluorescence and Microscopy Techniques
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The following primary antibodies were used against: EGFR (1005; Santa Cruz); ErbB2 (CB11; Serotec); ErbB3 (C17; Santa Cruz); α3-integrin (VM2; gift from F. Watt); E-cadherin (HECD1); α-catenin (VB1); β-catenin (VB2); β1-integrin (gift from F Giancotti); DOCK180 (C19 and H70; Santa Cruz); SOS1 (E1; Santa Cruz); actin (C4; MP Biomedicals); β-tubulin (Tub2.1; Sigma); Rac1 (23A8; Millipore, Upstate); RhoA (26C4; Santa Cruz); and Cdc42 (44; Transduction Laboratories). Secondary antibodies (fluorescent and HRP conjugates) were bought from DAKO.
Cells were fixed in 3% PFA for 10 min, washed in PBS and permeabilised with 0.1% Triton X-100 for 10 min at room temperature [23] (link). For E-cadherin and staining, keratinocytes on coverslips were imaged using a Leica DCS NT confocal microscope with Leica LCS Lite software. Z-slices of 0.2 μm were taken. Z-slices were summed and images were processed using open source ImageJ software.
For aggregation assays, phase contrast images were taken before and after mechanical trituration of aggregates using an Olympus CKX41 microscope and a Colour View IIIu camera linked to Sort Imaging System software. For bead assays, images were collected using a Zeiss Axiovert 200 microscope with Velocity software.
Cells were fixed in 3% PFA for 10 min, washed in PBS and permeabilised with 0.1% Triton X-100 for 10 min at room temperature [23] (link). For E-cadherin and staining, keratinocytes on coverslips were imaged using a Leica DCS NT confocal microscope with Leica LCS Lite software. Z-slices of 0.2 μm were taken. Z-slices were summed and images were processed using open source ImageJ software.
For aggregation assays, phase contrast images were taken before and after mechanical trituration of aggregates using an Olympus CKX41 microscope and a Colour View IIIu camera linked to Sort Imaging System software. For bead assays, images were collected using a Zeiss Axiovert 200 microscope with Velocity software.
6
Immunohistochemical Analysis of NSCLC Markers
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Tissue microarrays (TMAs) of NSCLCs and immunohistochemical staining of the five proteins were conducted as previously described [28 (link)]. The sections were incubated overnight at 4°C with the following primary antibodies: β-catenin (clone 17C2; Novocastra, Buckinghamshire, UK) at 1:100 dilution, c-MET (3D4, Zymed, South San Francisco, CA, USA) at 1:200 dilution, cyclin D1 (SP4, Lab Vision, Fremont, CA, USA) at 1:50 dilution, E-cadherin (4A2C7, Zymed, South San Francisco, CA) at 1:100 dilution, or EGFR (1005, Santa Cruz, CA, USA) at 1:100 dilution. Detection of immunoreactivity by each antibody was performed by EnvisionTM+ peroxidase (Dako, Carpinteria, CA, USA). The peroxidase activity for each antibody was visualized by applying diaminobenzidine chromogen containing 0.05% hydrogen peroxide for 3 to 8 min at room temperature. Nuclei were then counterstained with Mayer’s hematoxylin, and negative controls were included in each run by omitting the primary antibody.
7
Protein Expression Analysis in Cell Cultures
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Cells were seeded to 70% confluency in 24-well plates before treatments. Inhibitors were spiked into the media and incubated for the indicated times prior to cell lysis. Lysates were taken in boiling Laemmli buffer (125 mM Tris-HCl, 4% SDS, 0.01% bromophenol blue, 30% sucrose) with 0.05% β-mercaptoethanol (BME) and boiled for 5 minutes. Samples were analyzed by SDS-PAGE and blotted with the indicated primary antibodies: c-Met CVD13 (1:1000)(Invitrogen), integrin β4 H-101 (1:1000), Ron-β C-20 (1:750), and EGFR 1005 (1:750)(Santa Cruz Biotechnology, Santa Cruz, CA,), and β-tubulin (Neomarkers, Fremont, CA, 1:5000). High sensitivity streptavidin-HRP was obtained from Pierce Thermo Scientific (Rockford, IL, 1:10000). Blots were subsequently probed with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, Pittsburgh, PA), and detection was acquired with ECL (Amersham Biosciences).
8
Antibody Preparation and Detection
9
Epidermal Growth Factor Receptor Trafficking
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Epidermal growth factor (EGF) was purchased from Calbiochem, xanthine and bafilomycin A1 were purchased from Sigma. xanthine oxidase and leupeptin were obtained from Roche and 4', 6-diamidino-2-phenylindole (DAPI) and ammonium chloride from Merck. The antibodies used in this work were: rabbit anti-EGF receptor (EGFR) (1005) (Santa Cruz Biotech; sc-03), anti-human CD63, anti-LAMP1 and antihuman LAMP2 (Developmental Studies Hybridoma Bank, University of Iowa; H5C6, H4A3 and H4B4, respectively), rabbit anti-cathepsin B (FL-339) (Santa Cruz Biotech; sc-13985), mouse monoclonal anti-human CD222 (Bio-Legend, clone MEM-238; 315902), mouse anti-early endosome antigen 1 (EEA1) (BD Biosciences; 610457), and mouse monoclonal anti-tubulin (Sigma; clone B-5-1-2; T5168). The secondary antibodies used for immunostaining were horseradish peroxidase-coupled anti-mouse (Vector; PI-2000) and anti-rabbit (Nordic; GAR/IgG(H+L)/PO) antibodies, and antibodies labeled with Alexa Fluor 488 (Thermo Fisher; A-21206) or 555 (Thermo Fisher; A-31570).
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