APPwt-overexpressing human embryonic kidney 293 cells were grown in Dulbecco’s modified Eagle's medium (DMEM)–fetal calf serum (FCS) (10%)–penicillin/streptomycin medium. Cells were plated in 6-well dishes and allowed to secrete for 8 h in Opti-MEM/FCS (1%) containing phosphoramidon (10 μM) to prevent Aβ degradation by neprilysin (28 (link), 52 (link)), with or without aminopeptidase M (pl250), APA (pl302), and DPP4 (p32/98) inhibitors at 5 μM. After media centrifugation, supernatants were completed with one-tenth of RIPA buffer (Tris 50 mM; pH 7.4, containing NaCl (150 mM), EDTA (1 mM), Triton X100 (1%), deoxycholate (0.5%), and SDS (0.1%)) and incubated overnight with a 100-fold dilution of FCA18 antibody (26 (link)) and protein A agarose beads (VWR). Beads were washed twice with RIPA buffer 1× and once with Tris (10 mM, pH 7.5) and subjected to Tris/tricine 16.5% polyacrylamide gels. Proteins were transferred onto nitrocellulose and incubated overnight with the 2H3 monoclonal antibody (anti-Aβ1-12 provided by Dr D. Schenk, San Francisco, CA) at a 1/1000 dilution. After secondary antibody incubation with a goat anti-mouse peroxidase-conjugated antibody (1/2000 dilution), chemiluminescence was recorded using LAS-3000 (Raytest) and quantifications were performed using the Multi Gauge software.
Las3000
Manufactured by Elysia raytest
Sourced in United States
The LAS3000 is a laboratory imaging system designed for the analysis of gel-based assays. It provides high-resolution imaging capabilities for a variety of applications, including Western blotting, Northern blotting, and protein gel electrophoresis.
Automatically generated - may contain errors
3 protocols using las3000
1
Quantification of Aβ Secretion in HEK293 Cells
2
Western Blot Analysis of Protein Expression
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Cells were harvested as previously described [13 (link)] and protein isolated according to Laemmli [15 (link)]. Fifty μg protein/lane were subjected to Western blot analysis as recently described [13 (link)]. Membranes were incubated over night at 4°C with the primary monoclonal antibodies (mAb) directed against succinate dehydrogenase complex subunit A (SDHA; Cell Signaling), HIF2α (Novus) and β-actin (Sigma-Aldrich), followed by incubation for 1 h with horseradish peroxidase-linked secondary antibody and developed using the ECL method. Chemoluminescence signals were detected using a CCD camera (LAS3000, Raytest). For quantification of the SDHA protein expression the respective area of the signal was integrated using an AIDA image analyser (Raytest) and subsequently normalized to β-actin.
3
ADAM17 Expression in HEK Cells
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HEK cell lysates were probed for ADAM17 pro-form and mature form expression as described before [31 ]. Briefly, lysates were subjected to SDS-PAGE and proteins were transferred to nitrocellulose membrane (Amersham, UK). Membranes were blocked with 5% (w/v) non-fat dry milk in tris buffered saline with 0.05% Tween for 1 h and probed overnight at 4 °C against ADAM17 (1 µg/mL, rabbit polyclonal antibodies against C-terminus, Millipore, Darmstadt, Germany), followed by incubation with a POD-coupled secondary antibodies (diluted 1:20.000). After addition of the chemiluminescent substrate (Perkin Elmer, Waltham, MA, USA), signals were recorded using a luminescent image analyzer LAS3000 and quantified using AIDA image analyzer v.4.27 (Elysia-raytest GmbH, Straubenhardt, Germany).
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