Primary mouse antibodies HM50.67A and HM43.16B, were raised against the external domain of the HER-2 and EGFR, respectively (15 (link)). Mouse MAB3481 (anti-HER-3), MAB11311 (anti-HER-4), anti-insulin like growth factor receptor I (IGF-IR) mAbs and anti-E-cadherin were purchased from R&D Systems (Abingdon, UK). Secondary FITC-conjugated rabbit anti-mouse mAb STAR9B was obtained from AbD Serotec (UK). Gemcitabine was acquired from Healthcare at Home (UK) while The irreversible pan-HER family blocker afatinib was developed by Boehringer Ingelheim (Austria) as previously described (16 ,17 (link)). OSI-774 was kindly provided by OSI-Phamarceutical (USA). Doxycycline, 5-fluorouracil (5-FU), oxaliplatin, and mouse anti-EGFR antibody were purchased from Sigma-Aldrich. Mouse antibodies against β-actin and vimentin as well as rabbit antibodies against AKT, Mitogen-activated protein kinase (MAPK), phospho-MAPK (Thr202/Tyr204), phospho AKT (S473), Signal transducer and activator of transcription 3 (STAT3), p-STAT3 (Y705), Src, p-Src (Y416), c-MET (mouse), p-MET (Y1234/1235), p-EGFR (Y1086, 1068, 1143, 1173, 1045), p-HER3 (Y1289), HER3, HER2 and p-HER2 (Y1221/1222) were purchased from Cell Signalling, UK. The mouse anti-p-IGF-IR (Y1161) antibody and STAT3 inhibitor Stattic were purchased from Santa Cruz Biotechnology Inc. (Insight Biotechnology, UK).
Anti e cadherin
Manufactured by R&D Systems
Sourced in United States
Anti-E-cadherin is a reagent used in cell biology research. It is an antibody that binds to the E-cadherin protein, which is involved in cell-cell adhesion. This product can be used to detect and analyze the expression of E-cadherin in cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti vimentin, by Cell Signaling Technology (3 mentions)
Anti vinculin, by Merck Group (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Anti rhoa, by Cell Signaling Technology (2 mentions)
Anti cdc42, by Cell Signaling Technology (2 mentions)
Anti pkcε, by Cell Signaling Technology (2 mentions)
Anti pkcε, by Santa Cruz Biotechnology (2 mentions)
Anti phospho smad2 3, by Cell Signaling Technology (2 mentions)
Anti phospho mypt1, by Cell Signaling Technology (2 mentions)
Anti pkcδ, by Cell Signaling Technology (2 mentions)
Anti β actin, by BD (2 mentions)
Anti α smooth muscle actin, by R&D Systems (2 mentions)
Anti nf κb p65 subunit antibody, by Abcam (2 mentions)
Anti mcp 1, by Abcam (2 mentions)
Anti vimentin, by Santa Cruz Biotechnology (2 mentions)
Anti vimentin, by Merck Group (2 mentions)
Alexa fluor 488 affinipure bovine anti goat igg, by Jackson ImmunoResearch (2 mentions)
Cy3 affinipure donkey anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Accutase, by STEMCELL (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
26 protocols using anti e cadherin
1
Antibody Characterization for HER Family Signaling
2
Western Blot Analysis of Key Signaling Proteins
3
Organoid Formation from Dissociated Cells
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To determine the origin of cells forming the organoids, cells dissociated from tissues as mentioned above were stained with anti-E-cadherin (cat no. AF748; R&D Systems, Minnesota, United States) or anti-NGFR (cat no. sc-13577; Santa Cruz Biotechnology, Dallas, TX, USA) and their secondary antibody, Alexa Fluor 488–labeled donkey anti-goat IgG (cat no. A-11055; Invitrogen) or goat anti-mouse IgG (cat no. A-32723; Invitrogen). For direct labeling, FITC-conjugated anti-CD44 (cat no. 555478; BD Biosciences, Heidelberg, Germany), PE-conjugated anti-ITGA6 (cat no. 12-0495-83; BioLegend, San Diego, CA, USA), PE/Cyanine7-conjugated anti-NGFR Antibody (cat no. 345110; BioLegend) and APC-conjugated anti-EpCAM (cat no. 130-113-260; Miltenyi Biotec, Bergisch Gladbach, Germany) antibodies were used (Table S2 ). They were individually sorted using an S3e Cell Sorter (Bio-Rad, Hercules, CA, USA) and cultured in Matrigel with TeM for 15 days.
4
Protein Expression Analysis in Cell Types
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Whole cell protein and nuclear protein preparation, as well as western blotting, were performed as we described previously [24 (link), 25 (link)]. For whole protein, the membrane was probed with primary antibodies of anti-E-cadherin (rabbit polyclonal, R&D System, 1:1000), anti-α-smooth muscle actin (SMA) (rabbit polyclonal, R&D System, 1:1000), anti-fibroblast specific protein (FSP)-1 (rabbit polyclonal, Abcam, 1:1000), anti-collagen I (rabbit polyclonal, Calbiochem, 1:1000) and anti-MCP-1 (rabbit polyclonal, Abcam, 1:500); for nuclear protein, the membrane was probed with anti-NF-κB-p65 subunit antibody (rabbit polyclonal, Abcam, 1:1000). The intensities of the blots were determined using an imaging analysis program (Image J, free download from http://rsbweb.nih.gov/ij/ ). The β-actin was used as internal control. The normalized values in different groups were averaged and expressed as fold change with the mean value of control group as 1.
5
Immunofluorescence Staining of Cell Markers
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Cells were fixed and stained according to published methods [17 (link)]. Treated cells were fixed and incubated with the following antibodies: anti-E-cadherin (R&D Systems, 1:500), anti-β-catenin (1:500, R&D Systems), anti-Zeb1 (1:200, Abcam), and F-actin (Cell Signaling). Following primary incubation, conjugation with FITC (Sigma Biochemicals), Cy3 (Sigma Biochemicals), or Alexa Fluor 594 (Invitrogen) secondary antibodies was then performed. The cells were imaged with a Zeiss Axiophot microscope. Images were merged and analyzed using NIH Image J software.
6
Immunofluorescence Visualization of Epithelial-Mesenchymal Transition
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PANC-1, MIAPaCA-2 and BxPC-3 cells cultured in 35-mm µ-dishes (iBidi, Munich, Germany) were washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were then incubated with anti-E-cadherin (R&D Systems) and anti-vimentin (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h. Subsequently, cells were incubated with fluorescence-labeled secondary antibodies (Alexa Fluor 594, Life Technologies, Tokyo, Japan) for 1 h at room temperature and staining was observed using epi-illumination on a laser scanning confocal microscope (Olympus, Tokyo, Japan).
7
Immunohistochemical Analysis of Xenograft Tumors
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Formalin-fixed and paraffin-embedded 6 μm-thick tissue sections from xenograft tumors were processed for immunohistochemistry analysis as per the standard protocol. Sections were stained with anti-Ki67 antibody (SP6; Abcam) using the peroxidase technique. The proliferation index (%) was determined by calculating the number of Ki67-positive cells relative to the total number of cells, which consisted of at least 1000 cells per field. Serial sections were stained with anti-E-cadherin (1:100 dilution; R&D Systems) and anti-vimentin (1:100 dilution; Sigma) and the immune complexes were detected using the Vectastain Elite ABC-HRP kit (Vector Laboratories, Burlingame, CA). The sections were counterstained with hematoxylin.
TUNEL staining was performed to measure apoptosis in tumor tissue sections using In Situ Apoptosis Detection Kit (Abcam) according to the manufacturer’s instructions. The apoptosis index (%) was determined by calculating the number of TUNEL-positive cells relative to the total number of cells, which consisted of at least 1000 cells per field.
TUNEL staining was performed to measure apoptosis in tumor tissue sections using In Situ Apoptosis Detection Kit (Abcam) according to the manufacturer’s instructions. The apoptosis index (%) was determined by calculating the number of TUNEL-positive cells relative to the total number of cells, which consisted of at least 1000 cells per field.
8
Analyzing Tight Junction Proteins
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Cell or tissue lysates was subjected to SDS-PAGE and Western blot analysis with the use of anti-claudin1 (LifeSpan BioSciences, WA, USA), anti-E-cadherin (R&D Systems, MN, USA), anti-ZO-1 and anti-occludin (Proteintech group, Wuhan, China), anti-DSG1 (Invitrogen, CA, USA), and anti-GAPDH (Bioss, Beijing, China) antibodies followed by horseradish peroxidase–electrochemiluminescence (HRP-ECL) detection (Millipore, MA, USA).
9
Protein Lysate Western Blotting
10
Immunophenotyping of Differentiated hiPSCs
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Differentiated hiPSC were dissociated by Accutase, then fixed and permeabilized with eBioscience Foxp3 fixation/perm kit (Invitrogen) according to manufacturer instructions. The cells were then incubated for 30 min at 4°C with a mouse monoclonal anti-K14 (1/500, Millipore), Alexa fluor 488 mouse anti-human Pax6 (1/20, BD) and anti-E-cadherin (1/100, R&D) antibodies diluted in 0.1% bovine serum albumin (BSA, Sigma) in PBS. As control for Pax6 staining, Alexa Fluor 488 IgG2a, (1/5, BD) was used. Then the cells were washed with 2% FBS in PBS(-) and incubated for 30 min at 4°C with Alexa Fluor 488 donkey anti-mouse antibody (1/700, Life Technologies) as secondary antibody for E-cadherin and K14. The cells were then rinsed twice with 2% FBS in PBS. Cells were labelled with FITC-anti human CD104 (Biolegend, 1/20) without fixation/permeabilization steps. Fluorescence intensities were analyzed on a FC 500 cytometer (Beckman coulter) flow cytometer with CXP software.
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