Antibodies against phospho-Smad2 (p-Smad2), p-Smad3, Smad2, Smad3, ART, p-AKT, p-p38, p38, p-Erk1/2, Erk1/2, ECAD, NCAD, and TAZ were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-TRPV4 primary antibody was purchased from Alomone Labs (Jerusalem, Israel). Antibodies against β-Actin and GAPDH were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-α-SMA, GSK2193874 (GSK219), GSK1016790A (GSK101), SD208, and A23187 (A23) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse and rabbit anti-goat IgG were purchased from Jackson ImmunoResearch (West Grove, PA, USA). TGFβ1 was purchased from R&D Systems (Minneapolis, MN, USA). Alexa Fluor 488/594 conjugated IgG and Prolong diamond DAPI were purchased from Thermo Fisher Scientific (Waltham, MA, USA). FLIPR Calcium 5 assay kit was purchased from Molecular devices (Sunnyvale, CA, USA). Easy coat hydrogels of various degrees of stiffnesses (0.5, 1, 8, 12, 25, and 50 kPa) were purchased from Matrigen Life Technologies (Brea, CA, USA). Catalog number of all reagents and antibodies are included in the supplementary table 1 .
E cad
Manufactured by Cell Signaling Technology
Sourced in United States
E-cad is a laboratory equipment product offered by Cell Signaling Technology. It functions as a tool for the detection and analysis of E-cadherin, a key cell-cell adhesion protein.
Automatically generated - may contain errors
Lab products found in correlation
N cad, by Cell Signaling Technology (8 mentions)
Smad2, by Cell Signaling Technology (5 mentions)
Snail, by Cell Signaling Technology (5 mentions)
Vimentin, by Cell Signaling Technology (5 mentions)
P smad2, by Cell Signaling Technology (4 mentions)
Smad3, by Cell Signaling Technology (4 mentions)
P akt, by Cell Signaling Technology (3 mentions)
P erk1 2, by Cell Signaling Technology (3 mentions)
P smad3, by Cell Signaling Technology (3 mentions)
α sma, by Cell Signaling Technology (3 mentions)
β actin, by Merck Group (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Gapdh, by Thermo Fisher Scientific (3 mentions)
Gapdh, by Abcam (3 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
Stat3, by Cell Signaling Technology (2 mentions)
Cyclin d1, by Cell Signaling Technology (2 mentions)
N cad, by Abcam (2 mentions)
Horseradish peroxidase hrp labelled secondary antibody, by Beyotime (2 mentions)
Tgf β1, by Cell Signaling Technology (2 mentions)
36 protocols using e cad
1
Investigating TGF-beta Signaling Pathways
2
Immunofluorescence Staining of Tissue Samples
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Tissues were fixed in 4% paraformaldehyde at 4°C for 5 h before dehydration and then embedded in O.C.T for the frozen slice. Tissues were permeabilized with 0.2% Triton X100 in PBS for 10 min followed by blocking with 10% donkey serum in PBS for 1 h at room temperature. Primary antibodies were applied overnight at 4°C, followed by incubation with corresponding secondary antibodies-conjugated with Alexa Fluor 488 or 546 at room temperature for 1 h on the next day. The primary antibodies we used in the study are NANOG (R&D, AF1997), PAX6 (Biolegend, 901302), ECAD (Cell Signaling, 3195T), NCAD (Biolegend, 350802), SOX2 (R&D, AF12018), OCT4 (Abcam, ab181557), Nestin (Santa Cruz, sc-23927), PHH3 (Millipore, MABE941), ZO-1 (Thermo Fisher, 61-7300), PKCλ (BD, 610207), EZRIN (Sigma, E8897), PAX3(R&D, MAB2457), and NKX2.1(Santa Cruz, sc-13040). The stained coverslips were mounted for confocal laser scanning microscopy. We randomly select different positions for fluorescence intensity measurement with ZEN and performed intensity analysis with GraphPad Prism 7.
3
Western Blot Analysis of Cell Lysates
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The cells were collected and lysed with cell lysis buffer for the Western blot analysis. The proteins in the lysates (20 μg per lane) were separated on 8% or 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking with 5% nonfat milk in Tris-buffered saline with Tween 20 (TBST), the primary antibodies for FAP (R&D), FGF9 (R&D), E-cad (Cell Signaling Technology, MA, USA), N-cad (Cell Signaling Technology), Snail (Cell Signaling Technology), and GAPDH (Beyotime, Shanghai, China) were used overnight at 4 °C. The membranes were then washed with TBST three times and incubated with appropriate secondary antibodies. The protein bands were visualized using the enhanced chemiluminescence reagents (Millipore, MA, USA).
4
Protein Extraction and Analysis
5
Immunohistochemical Evaluation of EMT Markers
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Immunohistochemical (IHC) staining and evaluation were performed as previously described 9 (link). Antibodies against MEDAG (Biorbyt, orb380371), E-cadherin (E-cad; Cell Signaling Technology, 14472), N-cadherin (N-cad; Cell Signaling Technology, 13116) and Ki67 (Santa Cruz, sc-23900) were used.
6
NRG-Induced Fibroblast Activation Assay
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NRG was purchased from Sigma-Aldrich (CAS No. 71162; purity ≥95%). Dulbecco’s modified eagle medium and 0.25% Trypsin-EDTA were purchased from Gibco. Penicillin–streptomycin was purchased from BasalMedia. Fetal bovine serum was purchased from EXcell Bio. Dimethyl sulfoxide (DMSO) was purchased from Sevier Bio. Cell Counting Kit-8 (CCK-8) was purchased from Yeasen Biotechnology (Shanghai, China). TGF-β1 was purchased from GenScript Inc. Radioimmunoprecipitation assay (RIPA) buffer, phenylmethylsulfonyl fluoride, 50× cocktail protease inhibitor, phosphorylated protease inhibitor, and BCA protein quantitative detection kit were purchased from Servicebio. α-SMA, E-cad, phosphatidylinositol 3-kinase (PI3K), AKT, and p-Akt were purchased from Cell Signaling Technology. COLI was purchased from Affinity Biosciences. Fn was purchased from Abcam. Horseradish peroxidase goat anti-rabbit IgG (H + L) was purchased from ABclonal. The PAGE Gel Fast Preparation Kit was purchased from Epizyme. GAPDH, a Marker (10–180 kDa), and Super ECL Plus were purchased from PROTEINBIO.
7
Cellular Signaling Pathway Analysis
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Antibodies that recognize Cyclin D1 (Cell Signaling Technology, #2922), CDK4 (Cell Signaling Technology, #12790), β-actin (Sigma, A1978), NCAD (Abcam, ab98952), ECAD (Cell Signaling Technology, #3195), FOXO3 (Abcam, ab12162) and CTNNB1 (Ser33/37/Thr41) (Cell Signaling Technology, #4270) were used in the study. Horseradish peroxidase (HRP) labelled secondary antibody (Beyotime Biotech) was used in western blots. PS341 was purchased from Santa Cruz, sc-217785.
8
Immunohistochemical Analysis of Protein Expression
9
Western Blot Analysis of EMT Markers
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Cells and human RCC tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Beijing, China), supplemented with protease inhibitors (Roche, Shanghai, China) and the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF; Roche), at 4°C for 30min. The cell supernatants were extracted after centrifugation for 15 min at 14,000 rpm, then the concentration of the protein was determined using a BCA Protein Quantification kit (Beyotime Institute of Biotechnology). Proteins from tissues and cells were separated using 10% SDS-PAGE, transferred onto PVDF membranes (Millipore, Billerica, USA), blocked for 2h with 5% nonfat milk at room temperature, and incubated with primary antibodies at 4°C overnight. After that, the membranes were washed three times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Blots were detected using a Bio-Rad Bioimaging system (Bio-Rad, CA, USA), and antibodies against β-actin served as a negative control. Rabbit monoclonal antibodies (1:1000) against TGF-β1, E-cad, N-cad, Vimentin and MMP-9 (Cell Signaling Technology, USA) were used in Western blot analysis according to the manufacturer’s instructions.
10
Western Blot, qRT-PCR, and Flow Cytometry
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Western blot analysis was carried out as previously described 7 (link), 15 (link), 20 (link), 51 (link). Primary antibodies used are as follows: BRCA1, HSP90, GAPDH (Ambion), E-Cad, Vim, GATA3 (D13C9), (Cell Signaling), TWIST (Abcam). For qRT-PCR, total RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer's protocol and cDNA was generated using the Omniscript RT Kit (Qiagen). qRT-PCR was performed as previously reported 7 (link). For surface marker analysis, tumor cells were isolated and stained. After exclusion of lymphocytes, dead cells, and Lin+ cells, the expression profile for CD24 and CD29 was determined by flow cytometry as previously described 15 (link), 20 (link).
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