Alphafold was used to predict an interaction between iRhom2 and HLA, as it was previously used to model the iRhom2/ADAM17 interaction [29 (link)]. For the co-immunoprecipitation of iRhom2 and HLA, 2.2 × 106 cells were seeded in a 10 cm dish. Then, 10 µg of pcDNA3.1 plasmid containing HA tagged iRhom2, HA tagged inactive signal peptide peptidase-like 2 protease b (Sppl2b-D/A) or empty plasmid were transfected with 30 µl of Lipofectamine 3000 (Invitrogen). After 48 h, cells were lysed in 1 ml of lysis buffer (50 mm Hepes pH 7.4, 150 mm NaCl, 5 mm EGTA, 1% Glycerol, 1% Triton X100, 1.5 mm MgCl2, 10 mm 1,10-Phenanthroline) supplemented with complete protease inhibitor (Roche, Basel, CH). Cell lysates were cleared by centrifugation at 16,000 g for 20 min at 4 °C. 500 µl of cleared lysates were applied to agarose beads coupled with anti-HA antibodies (A2095, Sigma–Aldrich, US) and incubated o/n at 4 °C. Then, beads were washed 6 times in lysis buffer and incubated with 20 µl of Laemmli buffer at 65 °C for 20 min. Eluted proteins were loaded onto an acrylamide gel, separated by SDS-PAGE electrophoresis and then analysed by Western blotting using following antibodies: anti-ADAM17 (ab39162, Abcam, Cambridge, UK), anti-HA [HA7] (Sigma-Aldrich, St. Louis, MO, US), anti-HLA,ABC [EMR8-5] (ab70328, Abcam, Cambridge, UK) and anti-GAPDH (Code 5174, Cell Signaling, Danvers, Massachussets, US).
Anti adam17
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-ADAM17 is a laboratory reagent used for the detection and study of the ADAM17 protein. ADAM17, also known as tumor necrosis factor-alpha converting enzyme (TACE), is a member of the ADAM (A Disintegrin And Metalloprotease) family of proteins involved in the proteolytic processing of various cell surface proteins. Anti-ADAM17 can be used in techniques such as Western blotting, immunoprecipitation, and immunohistochemistry to identify and investigate the ADAM17 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti adam10, by Abcam (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Sorafenib, by Cell Signaling Technology (2 mentions)
Salirasib, by Merck Group (2 mentions)
Pf573228, by Bio-Techne (2 mentions)
Anti rabbit and anti mouse igg hrp linked antibodies, by Cell Signaling Technology (2 mentions)
Gefitinib, by Selleck Chemicals (2 mentions)
Fak antibody sampler kit, by Cell Signaling Technology (2 mentions)
Phospho egf receptor antibody sampler kit, by Cell Signaling Technology (2 mentions)
Phospho erk1 2 pathway sampler kit, by Cell Signaling Technology (2 mentions)
Alpha tubulin dm1a antibody, by Cell Signaling Technology (2 mentions)
Gi254023x, by Bio-Techne (2 mentions)
U0126, by Cell Signaling Technology (2 mentions)
Ir dye coupled secondary antibodies, by LI COR (2 mentions)
Gw280264x, by Aobious (2 mentions)
Fv1000, by Olympus (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Ab39162, by Abcam (1 mentions)
18 protocols using anti adam17
1
Immunoprecipitation of iRhom2 and HLA
2
Quantification of RNA and Protein Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using TRIzol reagent (Invitrogen) and quantified by NanoDrop-100 spectrophotometer. Real-time quantitative PCR was performed with an iQ5 (Bio-Rad Laboratories) [8 (link)]. TaqMan quantitative PCR assays (Applied Biosystems, Life Technologies) were run using primers/probes: TNF (Mm00443258 or Hs01113624), ADAM10 (Mm00545742 or Hs00153853), ADAM17 (Mm00456428 or Hs01041915), 18s (Mm03928990), and GAPDH (Hs02758991). Fold variation analyzed using delta-delta Ct (∆∆Ct) formula [18 (link)].
Proteins were assessed by western blot [8 (link)] using rabbit-anti-β-actin peroxidase (Sigma-Aldrich) or anti-ADAM17 (Abcam) followed by HRP-anti-rabbit IgG and detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).
Proteins were assessed by western blot [8 (link)] using rabbit-anti-β-actin peroxidase (Sigma-Aldrich) or anti-ADAM17 (Abcam) followed by HRP-anti-rabbit IgG and detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).
3
ADAM17-mediated Signaling Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells of each LEC subline (3×105) were lysed on ice in RIPA buffer containing 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% SDS, 1% NP-40 and Complete Protease Inhibitor Cocktail (Roche Applied Science). Western blotting analysis was performed according to a standard protocol [28 ]. Membranes were probed with the following antibodies at the indicated dilutions: rabbit polyclonal anti-ADAM17 (Abcam), 1:5,000; mouse monoclonal anti-BIG-H3 (Proteintech), 1:1,000; mouse monoclonal anti HB-EGF (MyBioSource), 1.5 μg/ml; rabbit monoclonal anti-EGFR (EP38Y) (Abcam), 1:100,000; rabbit polyclonal anti-ErbB2 (Abcam), 1:1,000; and mouse monoclonal anti-β-actin (Biodesign Int.), 1:5,000. Appropriate HRP-conjugated secondary antibodies (Sigma-Aldrich) were used. Bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore).
4
Raft Isolation and Immunoblot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extract preparation, immunoprecipitation assay, and sucrose gradient for raft isolation were performed as described elsewhere [28] (link). For immunoblot analysis, protein extracts were separated by NuPage 4% to 12% Bis-Tris gels (Life Technologies, Invitrogen), transferred to a nitrocellulose membrane, and probed with the following primary antibodies: anti–α-tubulin (Santa Cruz Biotechnology), anti-p56Lck (Santa Cruz Biotechnology), anti-Notch3 M20 (Santa Cruz Biotechnology), anti–lamin B (Santa Cruz Biotechnology), anti-HA (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Jagged1 C20 (Santa Cruz Biotechnology), anti–β-actin (Sigma-Aldrich), anti–c-Myc (Sigma-Aldrich) and anti-Flag (Sigma-Aldrich), anti-ADAM17 (Abcam, Cambridge, MA, USA), anti-Jagged1 N-terminal (Abcam), and anti–RBP-Jκ (Cell Signaling Technology, Beverly, MA, USA). Bound antibodies were detected with enhanced chemiluminescence (ECL kit, Amersham, GE Healthcare, Lafayette, CO, USA).
5
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To understand changes at the protein level, whole cell lysates prepared in radioimmunoprecipitation assay (RIPA) buffer were subjected to SDS-PAGE and western blotting, as described elsewhere. The following antibodies were used in this study: anti-claudin-5, anti-ADAM17 (Abcam, Cambridge, UK), anti-CD144, anti-β-catenin (Genetex, Irvine, CA), anti-occludin (Proteintech, Chicago, IL), anti-ZO-1 (Invitrogen, Waltham, MA), and anti-β-actin (Sigma-Aldrich, Chicago, IL). Densitometry of protein bands was performed using the Gel-Pro Analyzer software (Media Cybernetics, Rockville, MD, USA). β-actin was used as the loading control.
6
ADAM17 Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Annexin V-Alexa 568 and Hoechst 33342 were purchased from Thermo Fisher Scientific. OPS and Phorbol 12‐myristate‐13‐acetate (PMA) were obtained from Sigma-Aldrich. MM was purchased from Tocris Bioscience. anti-ADAM17 antibodies used for Western blotting were from Abcam (ab57484) and Merck Millipore (AB19027). Additional antibodies used were tubulin (clone E7, DSHB), AKT (serine/threonine kinase 1/protein kinase B; Cell Signaling Technology), and EGFR (Cell Signaling Technology). Peroxidase-coupled secondary antibodies for immunoblot were from Jackson Immunoresearch. Anti-actin was from Santa Cruz. Antibodies used for FACS analyses were anti-ADAM17 from Abcam and secondary anti-mouse Alexa Fluor 488 from Invitrogen.
7
Characterization of EGFR Signaling Inhibitors
8
Antibodies and Reagents Used in Study
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used in this study: antisera for TRAPα, Sec61β, GFP, and RFP were as described previously [[27] (link), [28] (link), [29] (link)]. Purchased antibodies included anti-FLAG M1 antibody (Merck, SF3040), anti-ADAM10 (Merck, AB19026), anti-ADAM17 (Abcam, ab2051), anti-GAPDH (Santa Cruz, SC-20357), anti-HA high affinity (Merck, 11867431001), anti-CD230 (prion) clone 3F4 (BioLegend, 800,307) and HRP-conjugated streptavidin (ThermoFisher, N100). The FLAG-conjugated GFP nanobody (GFP-Nb) was obtained as a gift from Ramanujan S. Hegde, MRC, UK [19 ].
Endo H, PNGase F, and all enzymes used for cloning were obtained from New England Biolabs. MG132, DTT, cycloheximide, thapsigargin and all chemicals used for the biochemical procedures in this study were purchased from Merck. Monensin and bafilomycin-A1 were purchased from MedChemExpress.
Endo H, PNGase F, and all enzymes used for cloning were obtained from New England Biolabs. MG132, DTT, cycloheximide, thapsigargin and all chemicals used for the biochemical procedures in this study were purchased from Merck. Monensin and bafilomycin-A1 were purchased from MedChemExpress.
9
Protein Expression Analysis in APPsw-Transfected HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
APPsw-transfected HeLa cells were washed with PBS and homogenized using lysis buffer (100 mM NaCl, 1% Triton X-100,1 mM sodium orthovanadate, protease inhibitors, 50 mM HEPES, pH 7.2). Centrifugation of cell lysates was carried out at 10,000× g for 10 min. The Bradford assay (Bio-Rad, Hercules, CA, USA) was used to measure protein concentration in the supernatant. SDS-PAGE (8% or 15%) was used to resolve proteins. The resolved proteins were transferred to a polyvinylidene fluoride membrane. Then, it was blocked using nonfat milk powder (5%) in Tris-buffered saline/Tween 20 (TBST) for 1 h at room temperature. The membrane was then incubated overnight at 4 °C with anti-sAPPα (Covance), anti-CTF (Sigma), anti-ADAM9 (Cell Signaling Technology, Danvers, MA, USA), anti-ADAM10 (Abcam), anti-ADAM17 (Abcam), anti-β-tubulin (Sigma), or β-actin (EnoGene, New York, USA). After washing membranes using TBST, horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (Cell Signaling Technology) were incubated at room temperature for 1 h. Enhanced chemiluminescence was performed to visualize the peroxidase activity. Multi Gauge Software (Fujifilm, Tokyo, Japan) using a LAS-4000 system was used to quantify the detected signals.
10
Immunostaining of Retinal ADAM17
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunostaining, retinas were dissected similarly as above. Retinas were fixed in 4% paraformaldehyde (VWR, 43368.9M) in PBS for 15 min and washed three times in PBS1X‐triton 0.3%. Retinas were labelled with anti‐ADAM17 (Abcam) overnight at 4°C followed by secondary antibody and phalloidin staining (Life Technologies, A12380) for 2 h at room temperature. Retinas were mounted in Vectashield (Vector Laboratories Ltd, H‐1000) and imaged on Olympus FV1000.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!