Snail clone c15d3

Manufactured by Cell Signaling Technology

Sourced in United Kingdom, United States

Snail (clone C15D3) is a laboratory reagent produced by Cell Signaling Technology. It is a monoclonal antibody that recognizes the Snail protein, a transcription factor involved in cellular processes.

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Lab products found in correlation

Vimentin clone d21h3, by Cell Signaling Technology (2 mentions) Ecl western blot detection system, by Cytiva (1 mentions) Ab80892, by Abcam (1 mentions) E cadherin clone hecd 1, by Abcam (1 mentions) Stat3, by Cell Signaling Technology (1 mentions) Trans blot turbo blotting system, by Bio-Rad (1 mentions) Mini protean tgx 4 15 gel, by Bio-Rad (1 mentions) Gapdh clone d16h11, by Cell Signaling Technology (1 mentions) Pierce ripa buffer, by Thermo Fisher Scientific (1 mentions) Imagequant las 4000 mini system, by GE Healthcare (1 mentions) Clarity western ecl substrate, by Bio-Rad (1 mentions) Edta free protease inhibitor cocktail, by Roche (1 mentions) Enhanced chemiluminescence detection kit, by Merck Group (1 mentions) Polyvinylidene difluoride membrane, by Merck Group (1 mentions) Foxo1 clone c29h4, by Cell Signaling Technology (1 mentions) Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions) Dc protein assay, by Bio-Rad (1 mentions) H3k27ac, by Cell Signaling Technology (1 mentions) H3k4me1, by Cell Signaling Technology (1 mentions) Clone 36 e cadherin, by BD (1 mentions)

4 protocols using snail clone c15d3

Western Blot Analysis of Epithelial-Mesenchymal Markers

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Whole cell lysates were prepared as previously described (21 (link)). Lysates (50 μg) were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. The membranes were then incubated with primary antibodies specific to Nanog (ab80892; Abcam, Cambridge, UK), Snail (clone C15D3, #3879; Cell Signaling Technology Japan, Tokyo, Japan), E-cadherin (clone HECD-1, ab1416), vimentin (ab24535) (both from Abcam), Stat3-phosphoTyr705 (clone D3A7, #9145), or Stat3 (#9132) (Cell Signaling Technology Japan), followed by peroxidase-conjugated IgG antibodies (#330 and #458; MBL, Nagoya, Japan). Anti-tubulin antibody was used to assess the levels of protein loaded per lane (clone DM1A, 05-829; Oncogene Research Products, Cambridge, MA, USA). Immune complexes were visualized using an ECL western blot detection system (Amersham, Aylesbury, UK).

Kusuoka O., Fujiwara-Tani R., Nakashima C., Fujii K., Ohmori H., Mori T., Kishi S., Miyagawa Y., Goto K., Kawahara I, & Kuniyasu H. (2017). Intermittent calorie restriction enhances epithelial-mesenchymal transition through the alteration of energy metabolism in a mouse tumor model. International Journal of Oncology, 52(2), 413-423.

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Western Blot Analysis of EMT Markers

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Protein was extracted from the cells using Pierce RIPA Buffer (Thermo Fisher Scientific, Rockford, IL, USA) with the cOmplete, EDTA‐free Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany). A total of 20 μg whole cell extracts was loaded on mini protean TGX 4–15% gels (Bio‐Rad, Hercules, CA, USA) and transferred using the Trans‐Blot Turbo Blotting System (Bio‐Rad). The membranes were probed with the following primary antibodies: mAbs for E‐cadherin (clone 24E10; Cell Signaling Technology, Beverly, MA, USA), vimentin (clone D21H3; Cell Signaling Technology), Snail (clone C15D3; Cell Signaling Technology), SERPINI1 (clone 1D10; Sigma‐Aldrich), CHST11 (clone 1H3; Sigma‐Aldrich), or GAPDH (clone D16H11; Cell Signaling Technology) as a control at 4°C overnight. The secondary antibodies were peroxidase‐coupled goat anti‐rabbit or anti‐mouse antibodies and detected with Clarity Western ECL Substrate (Bio‐Rad), and the protein bands were visualized using the ImageQuant LAS 4000 mini system (GE Healthcare Life Sciences).

Matsuda Y., Miura K., Yamane J., Shima H., Fujibuchi W., Ishida K., Fujishima F., Ohnuma S., Sasaki H., Nagao M., Tanaka N., Satoh K., Naitoh T, & Unno M. (2016). SERPINI1 regulates epithelial–mesenchymal transition in an orthotopic implantation model of colorectal cancer. Cancer Science, 107(5), 619-628.

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Immunoblotting protocol for protein analysis

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Immunoblotting was performed as described previously (14 (link)). Whole cell lysates were extracted from cells using Laemmli lysis buffer (12.5 mM Na₂HPO₄, 15% glycerol, 3% sodium dodecyl sulphate). The protein concentration was measured with the DC Protein Assay (BIO-RAD) and 30 μg of total protein was loaded onto 7.5 or 11% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore). After 1 h of blocking with 5% bovine serum albumin or non-fat milk prepared in TBS-Tween buffer, the blots were incubated overnight at 4°C with antibodies against FOXO1 (clone C29H4, 1:1000, Cell Signaling Technologies, ref #2880), XRN1 (1:1000, Bethyl Laboratories, ref A-300-443A), E-CADHERIN (clone 4A2C7, 1:500, Invitrogen, ref 33–4000), SNAIL (clone C15D3, 1:1000, Cell Signaling Technologies, ref #3879), SLUG (clone A-7, 1:400, Santa Cruz Biotechnology, ref sc-166476), VIMENTIN (clone SP20, prediluted, 1:100, Abcam, ab27608) and b-ACTIN (clone AC-40, 1:10 000, Sidma-Aldrich, ref A3853) used as loading control. After 1 h of incubation with a horseradish peroxidase-conjugated secondary antibody (1:6000, Santa Cruz Biotechnologies), protein bands were visualized using an enhanced chemiluminescence detection kit (Millipore) with the imaging system, Syngene Pxi4 (Syngene).

Zangari J., Ilie M., Rouaud F., Signetti L., Ohanna M., Didier R., Roméo B., Goldoni D., Nottet N., Staedel C., Gal J., Mari B., Mograbi B., Hofman P, & Brest P. (2016). Rapid decay of engulfed extracellular miRNA by XRN1 exonuclease promotes transient epithelial-mesenchymal transition. Nucleic Acids Research, 45(7), 4131-4141.

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Evaluating EMT-related Protein Expression

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Cells were lysed with the RIPA buffer containing 25 mM Tris-HCL, pH 7.5, 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA and 1.0% NP-40 on ice. Samples were resolved by SDS-PAGE and transferred to PVDF membranes. The western blots were performed with antibodies against MLL3 (Polyclonal, Millipore Sigma, ABE1851, 1:1000), E-cadherin (clone 36, BD Biosciences, cat# 610182, 1:1000), Vimentin (clone D21H3, Cell Signaling Technology, cat# 5741S, 1:1000), Slug (clone C19G7, Cell Signaling Technology, cat#9585S, 1:1000), Snail (clone C15d3, Cell Signaling Technology, cat#3879S, 1:1000), Zeb1 (clone D80D3, Cell Signaling Technology, cat#3396S, 1:1000), Stat1 (clone D1K9Y, Cell Signaling Technology, cat#14994S, 1:1000), phosphor-Stat1(Tyr701) (clone 58D6, Cell Signaling Technology, cat#9167S,1:1000), β-actin (clone C4/actin, BD Biosciences, cat#612656, 1:10,000), H3K27ac (clone D5E4, Cell Signaling Technology, cat#8173S, 1:1000), H3K4me1 (clone D1A9, Cell Signaling Technology, cat#5326S, 1:1000), H3K4me3 (clone C42D8, Cell Signaling Technology, cat#9751S, 1:1000), or Histone H3 (clone 1B1B2, Cell Signaling Technology, cat#14269S, 1:10,000). Original scans of the western blot results are provided in the Source Data for individual figures.

Cui J., Zhang C., Lee J.E., Bartholdy B.A., Yang D., Liu Y., Erler P., Galbo PM J.r., Hodge D.Q., Huangfu D., Zheng D., Ge K, & Guo W. (2023). MLL3 Loss Drives Metastasis by Promoting a Hybrid Epithelial-Mesenchymal Transition State. Nature cell biology, 25(1), 145-158.

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