Cells were homogenized in ice cold lysis buffer containing proteinase and phosphatase inhibitor cocktail. The primary antibodies utilized in this study were as follows: OCT4 (sc-5279; Santa Cruz Biotechnology), Nanog (A3233 ABclonal), NKX2.5 (ab91196; Abcam), α-actinin (6487; Cell Signaling Technology), CX43 (3512 Cell Signaling Technology), GATA4 (5851; Cell Signaling Technology), MEF2C (5030; Cell Signaling Technology), cTnI (ab47003; Abcam), p-ERK (4370; Cell Signaling Technology), ERK (5013 Cell Signaling Technology), β-Catenin (8480; Cell Signaling Technology), p-cadherin (2189; Cell Signaling Technology), p-STAT3 (RT1490; HuaBio), STAT3 (ET1605; HuaBio), and GAPDH (5174; Cell Signaling Technology). Blots were developed using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA, USA).
P cadherin
Manufactured by Cell Signaling Technology
Sourced in United States
P-cadherin is a lab equipment product from Cell Signaling Technology. It is a cell adhesion protein that plays a role in cell-cell interactions and signaling.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by Cell Signaling Technology (2 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
α actinin, by Cell Signaling Technology (1 mentions)
Mef2c, by Cell Signaling Technology (1 mentions)
Gata4, by Cell Signaling Technology (1 mentions)
P stat3, by Huabio (1 mentions)
Nanog, by ABclonal (1 mentions)
Stat3, by Huabio (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Mouse anti β tubulin clone tub 2, by Merck Group (1 mentions)
Ob cadherin, by Cell Signaling Technology (1 mentions)
Goat anti rabbit, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Goat anti mouse, by Agilent Technologies (1 mentions)
Mouse anti syndecan 1 clone b a38, by Abcam (1 mentions)
Keratin 10, by Fortrea (1 mentions)
Mouse anti ha 11 clone 16b12, by Fortrea (1 mentions)
N cadherin, by Abcam (1 mentions)
6 protocols using p cadherin
1
Western Blot Analysis of Cardiac Markers
2
Immunofluorescence Antibody Protocol
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Monoclonal antibodies used in this study included rabbit anti-αSMA antibody (clone E184; Abcam), mouse anti–α-actinin (clone BM75.2; Sigma-Aldrich), mouse anti-HA.11 (clone 16B12; Covance), mouse anti–β-tubulin (clone TUB 2.1; Sigma-Aldrich), mouse anti-paxillin (clone 5H11; EMD Millipore), mouse anti–syndecan-1 (clone B-A38; Abcam), rat anti–syndecan-4 (clone KY/8.2; BD), mouse anti-p120 catenin (clone 6H11; Invitrogen), rat anti–E-cadherin (clone DECMA-1; Abcam), and rabbit anti-NFAT3 (clone 23E6; Cell Signaling Technology). Polyclonal antibodies used were rabbit antibodies against syndecan-4 (Abcam and LSBio), OB-cadherin (Cell Signaling Technology), N-cadherin (Abcam), P-cadherin (Cell Signaling Technology), keratin-10 (Covance), TRPC4 (Sigma-Aldrich), and TRPC7 (Sigma-Aldrich and EMD Millipore). Secondary antibodies included goat anti–rabbit, goat anti–mouse, and rabbit anti–rat IgG HRP-conjugated antibodies from Dako. Donkey and goat anti–mouse, goat anti–rabbit, and donkey and goat anti–rat IgG conjugated to Alexa Fluor 488, 568, or 647 were obtained from Molecular Probes. DAPI and Alexa Fluor 568– and 647–conjugated phalloidin were obtained from Molecular Probes.
3
Proximity Ligation Assay for Protein-Protein Interactions
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Cells were deposited on glass slides and fixed with methanol for 10 min. PLA was performed using the Duolink kit (Olink Bioscience, Sweden), according to manufacturer’s recommendations. The following combination of primary anti-human antibodies were used against: P-cadherin (dilution 1:50, rabbit polyclonal IgG, Cell Signaling), E-cadherin (dilution 1:100, mouse monoclonal IgG1, clone HECD-1, Takara Bio Inc., Shiga, Japan; or dilution 1:50, rabbit monoclonal IgG, Cell Signaling), p120ctn (dilution 1:100, mouse monoclonal IgG1, clone 98, BD Biosciences). The nuclei were counterstained with DAPI. Slides were analyzed with fluorescence microscopy (Zeiss Imager Z1 microscope) for visualization of bright fluorescent red signals consistent with protein-protein interaction events. Ten stacks per image were taken and a minimum of five fields per experiment were evaluated. The Blobfinder V3.2 free software (Centre for Image Analysis, Uppsala, Sweden) was used to quantify the number of blobs (or dots) present in each condition. For cell culture experiments, an average of blobs/cells of at least three independent experiments was performed and was normalized to the control condition. For paraffin embedded tumors, the specific number of blobs/cell was analysed. The respective negative controls were used in each experiment.
4
Comprehensive Antibody Analysis of Cell Markers
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The following antibodies were used in this study: CK14 (Leica Microsystems, Wetzlar, Germany, LL-002 and Abcam, Cambridge, UK, ab 15461), CK19 (Abcam, ab7754), Actin (Abcam ab8229), glyceraldehyde 3-phosphate dehydrogenase (Abcam, ab9484), p63 (Abcam, ab3239), E-cadherin (BD, BD610682), N-cadherin (BD, BD610921), P-cadherin (Cell Signaling (CS), Danvers, MA, USA, #2130), EGFR (CS#4267), EGFR pY1068 (CS#3777), EGFR pY1173 (CS#4407), HER2 (CS#2165) HER2 pY1221/1222 (CS#2243), Axl (CS#8661), Ki67 (Abcam, ab15580), Cleaved Caspase-3 (Abcam, ab2302) and Phalloidin (Life Technologies, A22283, A12379).
5
Western Blot Analysis of Protein Expression
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The total protein was quantified using a BCA kit (Beyotime, Shanghai, China) and the protein concentration was adjusted based on the BCA assay results. Then, the proteins were transferred to PVDF membranes. The membranes were then blocked for 2 h at 25°C with 5% non‐fat dry milk and in TBS with 0.1% Tween‐20, and incubated overnight at 4°C with TRIM16 primary antibodies (ab72129, 1:1,500; Abcam), cleaved caspase‐3 (#9664, 1:1,000; Cell Signaling), caspase‐3 (#9662, 1:1,500; Cell Signaling, Shanghai, China), vimentin (#5741, 1:1,000; Cell Signaling), nephrin (ab216341, 1:1,000; Abcam), P‐cadherin (#2189, 1:1,000; Cell Signaling), BAX (#2772, 1:1,1,500; Cell Signaling), Bcl‐2 (#3498, 1:1,000; Cell Signaling), E‐cadherin (#96743, 1:1,500; Cell Signaling), N‐cadherin (#84117, 1:1,500; Cell Signaling), and β‐actin (#4970, 1:2,000; Cell Signaling). Then, the membranes were incubated with the secondary antibody for 1 h at 25°C. Immunoreactive bands were visualized with ECL western blotting (WB) substrate (Thermo Scientific), and the protein band intensity was quantified with ImageJ software and normalized to the signal intensity of β‐actin.
6
Protein Expression Analysis by Western Blot
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Protein extraction and western blot analysis were performed as per the protocols described earlier. 15 Briefly, MUT-MCF-7 and hGH-MCF-7 cells were lysed with hot sample buffer containing 1% sodium dodecyl sulfate (SDS) and then centrifuged for 15 min at room temperature. Protein extracts were separated by 10% SDSpolyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF; Hybond C-Extra; Amersham Pharmacia, Aylesbury, UK). After blocking, membranes were incubated with primary anti-mouse monoclonal antibodies (NF-κB (1:1000), E-cadherin (1:1000), P-cadherin (1:1000), and matrix metalloproteinase-9 (MMP-9; 1:1000) obtained from Cell Signaling Technology, Beverly, MA, USA)) for 1 h. The membranes were then washed and incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:10,000; Upstate Biotechnology, Lake Placid, NY, USA).
Blots were stripped and reprobed using anti-actin antibody (Sigma Aldrich, St. Louis, MO, USA). Protein bands were detected by the Phototype horseradish peroxidase western blot detection system (Cell Signaling Technology).
Blots were stripped and reprobed using anti-actin antibody (Sigma Aldrich, St. Louis, MO, USA). Protein bands were detected by the Phototype horseradish peroxidase western blot detection system (Cell Signaling Technology).
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