Cell lysates were prepared using complete lysis buffer (EMD Millipore, San Diego, CA), with protease and phosphatase inhibitor cocktails (Roche Diagnostics, Indianapolis, IN). Protein quantification was performed using DC protein assay from Bio-Rad (Hercules, CA). Western blot analysis was performed as described previously (Al-Azayzih et al. 2015 (link); Sabbineni et al. 2019 (link)). Antibodies used include Akt1 (2938), N-cadherin (4061), phosphorylated p38-MAPK (9211), total p38-MAPK (9212), phosphorylated Smad2/3 (8828), total Smad2/3 (8685), FoxC2 (12974), phosphorylated β-catenin (9561), total β-catenin (8480) and GAPDH (2118) from Cell Signaling Technology (Danvers, MA). Anti-β-actin (A5441) was from Sigma (St. Louis, MO), anti-eNOS (610297) was from BD Pharmingen (San Diego, CA), anti-ALK-5 (ab31013) was purchased from Abcam (Cambridge, UK) and anti-ALK-1 (NBP1-90254) was obtained from Novus Biologicals. Anti-phosphorylated-Smad1/5/8 (AB3848-I) from Millipore Sigma and anti-Smad1/Smad5/Smad8 (SAB2702532) from Sigma-Aldrich. Band densitometry was performed using NIH Image J software (Bethesda, MD).
N cadherin 4061
Manufactured by Cell Signaling Technology
Sourced in United States
N-cadherin (4061) is a primary antibody that recognizes the N-cadherin protein, which is a calcium-dependent cell-cell adhesion glycoprotein. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Complete lysis buffer, by Merck Group (1 mentions)
Anti β actin a5441, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
E cadherin 3195, by Cell Signaling Technology (1 mentions)
Stat3 9132, by Cell Signaling Technology (1 mentions)
P stat3 9138, by Cell Signaling Technology (1 mentions)
Anti β actin antibody sc 47778, by Santa Cruz Biotechnology (1 mentions)
Mem per eukaryotic membrane protein extraction reagent kit, by Thermo Fisher Scientific (1 mentions)
Iκbα 4814, by Cell Signaling Technology (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Vimentin sc 6260, by Santa Cruz Biotechnology (1 mentions)
0.22 μm nc membranes, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
4 protocols using n cadherin 4061
1
Western Blot Analysis of Cell Signaling
2
Mucosal Protein Profiling via Western Blot
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Western blot analysis was carried out with standard methods on the mucosal protein lysates obtained from each mouse. Specific antibodies against p-Stat3 (#9138), Stat3 (#9132), p-Src (#6943), Src (#2123), N-Cadherin (#4061), E-Cadherin (#3195), arginase-1 (#9819), and iNOS (#2982) were purchased from Cell Signaling Technology (Beverly, MA). Anti-FOXP3 antibody (ab54501) was purchased from Abcam (Cambridge, UK) and anti-α-tubulin (sc-20357) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
3
Protein Extraction and Immunoblotting Protocols
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Procedures of protein extraction and Immunoblot were described earlier [41 (link)]. Nuclear and cytoplasmic proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Waltham, MA, USA), membrane protein was extracted using Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Thermo Scientific, Waltham, MA, USA). Primary antibodies against beclin-1 (BECN1, ab51031), HIF-1α (ab51608), GLUT-1 (ab115730) and Snail (ab180714) were purchased from Abcam Public Limited Co (Cambridge, MA, USA), antibodies against LC3 (12741), N-cadherin (4061), E-cadherin (3195), Vimentin (5741), EpCAM (3599), IκBα (4814), Phospho-IκBα (2859), NF-κB p65 (8242), HIF-2α (7096), Lamin A/C (4777) and Na, K-ATPase (3010) were products of Cell Signaling Technology, Inc. (CST, Beverly, MA, USA), anti-Twist-1 antibody (AF62301) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA), anti-β-actin antibody (sc-47778) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA).
4
Western Blot Analysis of EMT Markers
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Cells protein lysates were separated by 10% SDS-PAGE, transferred to 0.22 μm NC membranes (Sigma), and incubated with specific antibodies. Anti-CDH1 and SNAI1 (Bioworld Technology), anti-β-catenin, and N-cadherin (4061) (Cell Signaling Technology), Vimentin (sc-6260) (Santa Cruz Biotechnology), and anti-β-actin (Sigma-Aldrich) were used as controls. Protein detection was performed with Super Signal Chemiluminescence Substrate (Pierce).
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