Western blotting was performed as described earlier.7 (link) Membranes were probed with the following antibodies: M75 hybridoma medium (1:3 in 5% non-fat dry milk with 0.2% Nonidet P40 in PBS, 1 h, RT); anti-β-actin (Cell Signalling, 1:5000 in 3% BSA in TBS-T buffer, 1 h, RT); anti-ADAM17 (Santa Cruz Biotechnology, TX, USA, 1:300 in 3% BSA in TBS-T buffer, 2 h, RT); anti-IGFBP-3 (R&D Systems, MN, USA, 1:500 in 3% BSA in TBS-T buffer, overnight, 4 °C); anti-IGFBP2 (GeneTex, CA, USA, 1:1000 in 3% BSA in TBS-T buffer, overnight, 4 °C), anti-THBS-1 (Thermo Fisher Scientific, MA, USA, 1.5 µg/ml in 3% BSA in TBS-T buffer, 1 h, RT); anti-EMMPRIN/Basigin (Invitrogen, CA, USA, 1:250 in 3% BSA in TBS-T buffer, 2 h, RT); anti-mouse-HRP or anti-rabbit-HRP (Sigma-Aldrich, MO, USA, 1:5000 in 5% non-fat dry milk with 0.2% Nonidet P40/0.1% Tween20 in PBS, 1 h, RT). Protein bands were detected using enhanced chemiluminescence kit (GE Healthcare Bio-Sciences, IL, USA).
Anti adam17
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom
Anti-ADAM17 is a laboratory reagent that can be used to detect and quantify the expression of ADAM17, also known as tumor necrosis factor-alpha converting enzyme (TACE), in biological samples. ADAM17 is a member of the ADAM (a disintegrin and metalloproteinase) family of proteins and plays a role in the proteolytic processing of various cell surface proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti adam10, by Abcam (2 mentions)
Anti igfbp 3, by R&D Systems (1 mentions)
Enhanced chemiluminescence kit, by GE Healthcare (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti β actin hrp, by Merck Group (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Phospho stat3, by Cell Signaling Technology (1 mentions)
Anti akt, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti tnf α, by Santa Cruz Biotechnology (1 mentions)
Anti cldn5, by Thermo Fisher Scientific (1 mentions)
Tcs sp5 spectral confocal microscope, by Leica camera (1 mentions)
Affinipure goat anti rabbit igg h l, by Proteintech (1 mentions)
Anti il 18, by Affinity Biosciences (1 mentions)
Anti il 6, by Affinity Biosciences (1 mentions)
Anti tlr4, by Proteintech (1 mentions)
Anti il 1β, by ABclonal (1 mentions)
Anti caspase 1, by ABclonal (1 mentions)
Anti p38 mapk, by ABclonal (1 mentions)
Anti nf κb, by ABclonal (1 mentions)
6 protocols using anti adam17
1
Western Blotting Antibody Probing Protocol
2
Western Blot Analysis of Cell Signaling
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Cell lysates were prepared in RIPA buffer (50-mM Tris·HCl, pH 8, 150-mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 1% Triton X-100 plus proteinase inhibitors). Protein concentration was determined by Bradford assay, and samples containing 10–50 μg were separated by sodium dodecyl sulfate protein gel electrophoresis. Proteins were transferred to nitrocellulose membranes. After this, membranes were blocked in 8% nonfat milk in 20-mM Tris-HCl, 150-mM NaCl, and 0.05% Tween 20 for 1 hour at room temperature. Anti-AKT (sc-8312; Santa Cruz Biotechnology) anti-phospho-AKT (#9271; Cell Signaling); phospho-AXL (#PA5-39729; Invitrogen), phospho-MERTK (#SAB4504621; Sigma-Aldrich), phospho-STAT3 (#9145S; Cell Signaling), anti-ADAM10 (#ab1997; Abcam, Cambridge, United Kingdom), anti-ADAM17 (#sc-390859; Santa Cruz Biotechnology), anti-GAPDH (#ab181602; Abcam), and anti-β-actin-HRP (#A3854; Sigma-Aldrich).
3
Myocardial Infarction ADAM17 and TNF-α Analysis
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The blanched area of myocardial infarcted heart tissue was separated and fixed using 10% formalin buffer for 24 hours. The following actions were dehydration, clearing, impregnation, and blocking. Tissue was made of 6 μm thickness and immunohistochemistry was performed to see the expression of ADAM17 (anti-ADAM17, Santa Cruz Biotechnology, United States) and TNF-α (anti-TNF-α, Santa Cruz Biotechnology, United States). Samples were washed with PBS pH 7.4 and immunohistochemical staining was carried out according to the kit’s instruction (novoLink novocastra, Leica paint # RE7150-CE).
Resulted slides were observed using a Nikon E100 microscope. The measurements of each parameter (expression of ADAM17 and TNF-α) used a calculation technique of 20 fields with a magnification of 1000×, each containing approximately 1500 cells. The images were documented with a 400× magnification by using Panasonic GX-8 camera.
Resulted slides were observed using a Nikon E100 microscope. The measurements of each parameter (expression of ADAM17 and TNF-α) used a calculation technique of 20 fields with a magnification of 1000×, each containing approximately 1500 cells. The images were documented with a 400× magnification by using Panasonic GX-8 camera.
4
Immunofluorescence Analysis of ADAM17, GNAZ, and CLDN5
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Cells were grown on glass coverslips and illuminated with blue light. After the routine protocol involving PBS washing, ice-cold methanol fixation, and blocking, the samples were incubated overnight (4 °C) with primary antibodies, which included anti-ADAM17 (1:50 dilution; Santa Cruz, Dallas, TX, catalog no. sc-390859), anti-GNAZ (1:100 dilution; Cusabio, Houston, TX, catalog no. CSB-PA002856), and anti-CLDN5 (1:100 dilution; Invitrogen, Waltham, MA; catalog no. 34–1600). The samples were washed three times and incubated with a secondary antibody (1:1000) at room temperature for 1–2 h in the dark. Before mounting, the samples were incubated with DAPI for a short period of time to visualize the nuclei. Fluorescent images were captured using a Leica TCS SP5 confocal spectral microscope.
5
Investigating Inflammatory Signaling Pathways
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Santa Cruz Biotechnology (Dallas, Texas, USA) provided the anti‐TIMD4 (Cat# SC‐390805), anti‐ADAM17 (Cat# SC‐390859), and TGF‐β1 (Cat# sc‐130348) antibodies. Affinity Biosciences (Cincinnati, Ohio, USA) provided the anti‐p‐NF‐κB (Cat# AF2006), anti‐IL‐6 (Cat# DF6087), anti‐IL‐10 (Cat# DF6894), and anti‐IL‐18 (Cat# DF6252) antibodies. ABclonal (Boston, MA, USA) provided anti‐NF‐κB (Cat# A16271), anti‐phosphorylated p38 MAPK (p‐p38 MAPK, Cat# AP0526), anti‐p38 MAPK (Cat# A14401), anti‐caspase‐1 (Cat# A0964), and anti‐IL‐1β (Cat# A16288) antibodies. Proteintech (Chicago, IL, USA) provided the anti‐TLR‐4 (Cat# 66350‐1‐Ig), anti‐TGF‐β1 (Cat# 21898‐1‐AP) antibodies, HRP‐conjugated GAPDH monoclonal antibody (Cat# HRP‐60004), and fluorescein‐conjugated AffiniPure Goat Anti‐Rabbit IgG (H+L) (Cat# SA00003‐2). MedChemExpress (Monmouth Junction, NJ, USA) provided TAPI‐1 (Cat# HY‐16657) and SB203580 (Cat# HY‐10256). Yiyuan Biotechnology (Guangzhou, China) provided ox‐LDL (Cat# YB‐002). Sigma–Aldrich (St. Louis, MO, USA) provided LPS (Cat# L2630).
6
Immunodetection of ADAM10 and ADAM17
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Total cell lysates were obtained as described previously (2) . Immunodetection was performed by standard procedures, according to the manufacturer's guidelines (ECL; Amersham, Buckinghamshire, UK), using anti-ADAM10 (Abcam, Cambridge, UK), anti-ADAM17 and anti-ß-actin (both obtained from Santa Cruz Biotechnology).
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