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4 protocols using anti n cadherin d4r1h

1

Immunoblotting for Epithelial-Mesenchymal Markers

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Immunoblots were carried out according to standard methods [28 (link)] using rabbit antibody anti-N-Cadherin (D4R1H), anti-ZEB-1 (D80D3), anti-Vimentin (D21H3), anti-β-Catenin (D10A8), anti-Claudin-1 (D5H1D) (Cell Signaling Technology Inc., Danvers, MA, USA Upstate, Lake Placid, NY, USA) and anti-GPX4 (Abcam, Cambridge, UK). Anti-rabbit secondary antibodies coupled with horseradish peroxidase (Cell signaling Technologies) were utilized.
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2

Western Blot Analysis of EMT Markers

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Protein expression was analyzed by Western blot assays, as previously described [53 (link)]. At least three biological assays were performed. A summarized protocol is shown in the Supplementary Materials: Supplementary Methods/Western blot analysis. The following rabbit monoclonal antibodies were used in this study: anti-Vimentin (D21H3, #5741), anti-E-cadherin (24E10, #3195), anti-Snail (C15D3, #3879), anti-Slug (C19G7, #9585), anti-N-cadherin (D4R1H, #13116), and anti-β-Actin (13E5, #4970) from Cell Signaling Technologies Inc. (Danvers, MA, USA); anti-CDA (EPR20525, #ab222515) from Abcam (Cambridge, UK); and anti-Zeb1 (#HPA027524) from Sigma-Aldrich. HRP-conjugated goat anti-rabbit (#7074) secondary antibody was obtained from Cell Signaling Technologies Inc.
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Culturing CAL27 and SCC4 Cell Lines

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CAL27 and SCC4 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen, Germany), 2 mM L-glutamine (Invitrogen, Germany), and antibiotics (50 μg/ml penicillin–streptomycin, Invitrogen, Germany). Mouse anti-α-tubulin, rabbit anti-E-cadherin (24E10), and anti-N-cadherin (D4R1H) were from Cell Signaling Technology (Beverly, MA). Rabbit anti-Snail and anti-TGF-β1 were from Abcam (Cambridge, MA). Rabbit anti-HNRNPA2B1 was from ProteinTech (Rosemont, IL, USA), and mouse anti-ORF1p was purchased from Sigma-Aldrich (clone 4H1, Germany).
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4

Targeting Stat3 in Cancer Metastasis

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rhG-CSF protein and TGF-β1 peptide were purchased from Peprotech and R&D Systems, respectively. The Stat3 inhibitor Stattic (HY-13818) was obtained from Med Chem Express (MCE). G-CSF-neutralizing antibody (ab9691) was purchased from Abcam. Anti-Stat3 (D1B2J), anti-Stat3 (p-Tyr705) (D3A7), anti-vimentin (D21H3), and anti-N-cadherin (D4R1H) antibodies were obtained from Cell Signaling Technology. Anti-β-actin (60008-1-Ig), anti-CD44 (ab216647), and anti-CD90 (66766-1-Ig) antibodies were from Proteintech. The nonspecific (NS) and two Stat3-specific siRNAs, targeting GGCGTCCAGTTCACTACTA and AGACCCGTCAACAAATTAA, respectively, were purchased from RiboBio.
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