Adam17

Manufactured by Santa Cruz Biotechnology

Sourced in Germany

ADAM17 is a laboratory reagent used in biomedical research. It is a disintegrin and metalloproteinase domain-containing protein that functions as a sheddase, cleaving the extracellular domains of various membrane-bound proteins. ADAM17 is involved in the release of cytokines, growth factors, and other signaling molecules.

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Anti-ADAM17 (6 citations)

4 protocols using adam17

Western Blot Analysis of Signaling Proteins

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As described in our previous study (16 (link)), REC were rinsed with cold PBS, collected in lysis buffer containing protease and phosphatase inhibitors, and scraped into tubes. Equal amounts of protein were separated on precast tris-glycine gels (Invitrogen, Carlsbad, CA), and then blotted onto a nitrocellulose membrane. After blocking in TBST (10mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, the membrane was treated with appropriate primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were detected by chemilluminescence reagent kit (Thermo Scientific, Pittsburgh, PA). Primary antibodies used were phosphorylated STAT3 (Tyr705) and total STAT3 (purchased from Cell Signaling, Danvers, MA), VEGF (Abcam, Cambridge, MA), ADAM10, ADAM17 (Santa Cruz Biotechnology, Dallas, TX), and beta actin (Santa Cruz, Santa Cruz, CA).

Ye E.A, & Steinle J.J. (2017). miR-146a suppresses STAT3/VEGF pathways and reduces apoptosis through IL-6 signaling in primary human retinal microvascular endothelial cells in high glucose conditions. Vision research, 139, 15-22.

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Western Blotting of Protein Targets

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Cell or tissue lysates were lysed in 4% SDS buffer followed by needle homogenization. Equal amounts of protein (15-40 μg) were mixed with loading dye, boiled for 8 min, separated on a denaturing SDS-polyacrylamide gel and transferred to a PVDF membrane. The membrane was blocked in 5% milk/TBS/0.1% Tween for 1h and incubated with antibodies against ADAM17, NFκB, p21, GAPDH (Santa Cruz Biotechnology, Dallas, TX), Collagen IA (Southern Biotech, Birmingham, AL), TGFβ1 (abcam, Cambridge, MA), pSTAT3, pERK, pAKT (Cell Signaling, Danvers, MA), or ATGL (eBioSci, San Diego, CA).
The membrane was washed with TBS-0.1% Tween and then incubated with HRP-conjugated secondary antibody (Santa Cruz) at room temperature for 1h and rewashed. Protein bands were visualized by an enhanced chemiluminiscence method (Thermo, Waltham, MA) and resolved digitally per the manufacturer's specifications.

Principe D.R., DeCant B., Diaz A.M., Mangan R.J., Hwang R., Lowy A., Shetuni B.B., Sreekumar B.K., Chung C., Bentrem D.J., Munshi H.G., Jung B., Grippo P.J, & Bishehsari F. (2016). PEDF inhibits pancreatic tumorigenesis by attenuating the fibro-inflammatory reaction. Oncotarget, 7(19), 28218-28234.

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ADAM17 and ERK Protein Detection

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Cell suspensions were lysed in phosphate buffered saline/1% Triton X-100/protease inhibitor cocktail (Roche, Mannheim, Germany). 10 μg of was loaded on 7.5% standard polyacrylamide gels and transferred onto polyvinylidene fluoride PVDF membranes (all reagents from Peqlab, Erlangen, Germany). ADAM17 (Santa Cruz Biotech, Heidelberg, Germany) antibodies were diluted 1:500, and extracellular signal-regulated kinase/phosphorylated extracellular signal-regulated kinase antibodies (Cell Signaling, Frankfurt, Germany) were used at 1:600. Staining with anti-actin (1:500; Santa Cruz Biotechnology, Heidelberg, Germany) served as loading control. Blots were visualized by horseradish peroxidase-labeled secondary reagents (Dianova, Hamburg, Germany) and chemiluminescence (ECL; GE-Healthcare, Freiburg, Germany).

Mahnke K., Useliene J., Ring S., Kage P., Jendrossek V., Robson S.C., Bylaite-Bucinskiene M., Steinbrink K, & Enk A.H. (2016). Down-Regulation of CD62L Shedding in T Cells by CD39+ Regulatory T Cells Leads to Defective Sensitization in Contact Hypersensitivity Reactions. The Journal of investigative dermatology, 137(1), 106-114.

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Western Blot Analysis of Protein Targets

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Cell lysates were electrophoresed to resolve proteins by SDS-PAGE, transferred onto a nitro-cellulose membrane, and blocked with 4% non-fat dry milk. The membrane was incubated at 4°C overnight with specific primary antibody, followed by a secondary antibody conjugated with horseradish peroxidase. The protein bands were detected by chemiluminescence (Life Technologies). The blot from the same run was reprobed with β-actin (Sigma) or α-tubulin (Santa Cruz Biotechnology) HRP conjugated antibody to compare protein load in each lane. Commercially available antibodies for ACE2, AT1 receptor, c-Fos, IκBα, ADAM-17, MCP-1 and IL-6Rα were procured from Santa Cruz Biotechnology; phospho-p38 MAPK (Thr180/Tyr182), phospho-p42/44 MAPK (Thr202/Tyr204), phospho-NF-κB (Ser276), SOCS3, phospho-STAT3 (Tyr705), phospho-STAT3 (Ser727), total STAT3 were procured from Cell Signaling Technologies; and SARS-CoV-2 spike protein (Sino Biological) were also procured for western blot analyses.

Patra T., Meyer K., Geerling L., Isbell T.S., Hoft D.F., Brien J., Pinto A.K., Ray R.B, & Ray R. (2020). SARS-CoV-2 spike protein promotes IL-6 trans-signaling by activation of angiotensin II receptor signaling in epithelial cells. PLoS Pathogens, 16(12), e1009128.

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