Cst4970

Manufactured by Cell Signaling Technology

Sourced in Germany, United Kingdom

CST4970 is a laboratory equipment product offered by Cell Signaling Technology. It is a device designed to perform a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation.

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Lab products found in correlation

4 protocols using cst4970

Western Blot Analysis of Tight Junction and Inflammatory Proteins

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Colon tissues were lysed in 1 mL RIPA lysis buﬀer containing 1 mM PMSF and 1% phosphatase inhibitor cocktail (Applygen Technologies Inc., Beijing, China). Protein concentrations were measured using a Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Samples (25–50 μg) were separated using 10% SDS–PAGE and then wet transferred to polyvinylidene diﬂuoride membranes (PVDF, EMD Millipore, Burlington, USA). Membranes were blocked with 5% non-fat milk and incubated with primary antibodies against occludin (1:1000; ab167161, Abcam), claudin-1 (1:1000; ab180158, Abcam), ZO1 (1:1000; CST#13663,Cell Signaling Technologies, Darmstadt, Germany), TLR4 (1:500; sc-293072, Santa Cruz), NF-κB (1:1000; CST#8242, Cell Signaling Technologies), phosphorylated (p) NF-κB (1:1000; CST#8214, Cell Signaling Technologies), MyD88 (1:1000; ab2064, Abcam), or β-actin (1:1000; CST#4970, Cell Signaling Technologies) and incubated overnight at 4°C with gentle shaking. The membranes were washed three times and then incubated with a horseradish peroxidase–linked secondary antibody (1:1000; Beyotime, Jiangsu, China). Immunoreactivity was detected using an enhanced chemiluminescence reagent (WBKLS0500; EMD Millipore) and quantiﬁed using Image Lab 5.2.1 software (Bio-Rad, Hercules, CA, USA).

Ji C., Deng Y., Yang A., Lu Z., Chen Y., Liu X., Han L, & Zou C. (2020). Rhubarb Enema Improved Colon Mucosal Barrier Injury in 5/6 Nephrectomy Rats May Associate With Gut Microbiota Modification. Frontiers in Pharmacology, 11, 1092.

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Immunoblotting Analysis of Cellular Signaling

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Cells were homogenized in 100 μL Kaplan buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1% NP-40, 10% glycerol, and a protease inhibitor cocktail (Roche Diagnostics)). The lysate was subject to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and WB analysis with a standard procedure [62 (link)] using antibodies against AKT (1:1000; CST 4691; Cell Signaling Technology, Beverly, MA, USA), phospho-AKT (1:1000; CST 4056; Cell Signaling Technology), ERK1/2 (1:1000; CST 4695; Cell Signaling Technology), phospho-ERK1/2 (1:1000; CST 9101; Cell Signaling Technology), p38 (1:1000; CST 9212; Cell Signaling Technology), phospho-p38 (1:1000; CST 9215; Cell Signaling Technology), JNK (1:1000; CST 9252; Cell Signaling Technology), phospho-JNK (1:1000; CST 4668; Cell Signaling Technology), c-JUN (1:1000; CST 9165; Cell Signaling Technology), phospho-c-JUN (1:1000; CST 3270; Cell Signaling Technology), MBP (1:1000; M3821; Sigma-Aldrich), MAG (1:1000; MAB1567; Chemicon, Temecula, CA, USA), P0 (1:1000; ab31851; Abcam, Cambridge, UK), HIF1α (1:1000; CST 14179; Cell Signaling Technology), and β-actin (1:1000; CST 4970; Cell Signaling Technology). Protein expression levels were determined using the MF-ChemiBIS 3.2 imaging system (Berthold Technologies, Bad Wildbad, Germany).

Matsuoka H., Ebina K., Tanaka H., Hirao M., Iwahashi T., Noguchi T., Suzuki K., Nishimoto S., Murase T, & Yoshikawa H. (2018). Administration of Oxygen Ultra-Fine Bubbles Improves Nerve Dysfunction in a Rat Sciatic Nerve Crush Injury Model. International Journal of Molecular Sciences, 19(5), 1395.

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Western Blot Analysis of Apoptosis Regulators

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Proteins were separated by SDS-PAGE (Mini-Protean TGX Precast Gel 12%, 456-1045, Bio-Rad, Madrid, Spain) and transferred to PVDF membranes (10600023, Amersham Hybond, Pittsburgh, PA, USA). Membranes were blocked with dry milk dissolved in Tris-buffered saline with 1% Tween 20 (TBST) for 1 h and probed overnight at 4 °C with the primary antibodies of interest directed against rabbit anti-BCL-xL (CST2764, Cell Signaling, Leiden, The Netherlands), rabbit anti-MCL-1 (CST5453, Cell Signaling, Leiden, The Netherlands), rabbit anti-BIM (CST2933, Cell Signaling, Leiden, The Netherlands), rabbit anti-actin (CST4970, Cell Signaling, Leiden, The Netherlands) followed by anti-rabbit IgG HRP-linked secondary antibody (CST7074, Cell Signaling, Leiden, The Netherlands) in 3% BSA in TBST for 1 h at room temperature. Immunoblots were developed using Clarity ECL Western substrate (1705060, Bio-Rad, Madrid, Spain). When necessary, immunoblots were stripped in 0.1 M glycine pH 2.5, 2% SDS for 40 min, and washed in TBS. Bands were visualized with LAS4000 imager (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and ImageJ were then used to measure the integrated optical density of bands.

Alcon C., Zañudo J.G., Albert R., Wagle N., Scaltriti M., Letai A., Samitier J, & Montero J. (2021). ER+ Breast Cancer Strongly Depends on MCL-1 and BCL-xL Anti-Apoptotic Proteins. Cells, 10(7), 1659.

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Immunostaining and Western Blot Antibodies

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Antibodies against B220 (ab64100, 1:50 dilution), Igκ light chain (ab190484, 1:100 dilution), GFAP (ab7260, 1:1000 dilution), synaptophysin (ab32127, 1:400 dilution), HA-tag (ab24779, 1:1000 dilution), PCNA (ab29, 1:10,000 dilution), GD3 (ab11779, 1:1000 dilution), nestin (ab6142, 1:1000 dilution), olig2 (ab109186, 1:100 dilution), E-cadherin (for IHC, ab76055, 1:200 dilution), PCK (ab7753, 1:250 dilution), p53 (for IHC, ab31333, 1:50 dilution), phosphorylated-p53 Ser15 (for IHC, ab1431, 1:50 dilution), vimentin (ab92547, 1:200 dilution), NTP-II (ab33595, 1:250 dilution) and α-SMA (ab5694, 1:50 dilution) were purchased from Abcam (Cambridge, MA). Antibodies against p53 (for western blotting [WB], CST2524, 1:1000 dilution), phosphorylated-p53 Ser15 (for WB, CST9248, 1:1000 dilution), GAPDH (CST5174, 1:1000 dilution), and β-actin (CST4970, 1:5000 dilution) were from Cell Signaling Technology (Danvers, MA). Antibodies against β-casein (SC-166684, 1:20 dilution) and β-catenin (SC-7963, 1:50 dilution) were from Santa Cruz Biotechnology (Dallas, TX). miRNA mimics and anti-miRNAs for miR-382, miR-325, and their controls (mirVana miRNA Mimic Negative Control #1 and Anti-miR Negative Control #1) were purchased from ThermoFisher Scientific (Waltham, MA). Lentiviral vectors expressing miRNA mimics and anti-miRNAs for miR-382, miR-325, and control miRNA were purchased from Sigma-Aldrich.

Deng Q., Hu H., Yu X., Liu S., Wang L., Chen W., Zhang C., Zeng Z., Cao Y., Xu-Monette Z.Y., Li L., Zhang M., Rosenfeld S., Bao S., Hsi E., Young K.H., Lu Z, & Li Y. (2019). Tissue-specific microRNA expression alters cancer susceptibility conferred by a TP53 noncoding variant. Nature Communications, 10, 5061.

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