Control or RPE1 cells stably expressing a small hairpin RNA targeting CMG2 mRNA were seeded on plates and incubated overnight in DMEM complemented with Gropro (Zen-Bio), to remove the collagen VI present in FBS. The next days, cells were incubated or not for 60 min with 100 nM bafilomycin (Sigma). Thirty minutes before the addition of purified collagen VI tetramer (kind gift from P. Bonaldo), 5% BSA was added to the cell culture medium to block aspecific binding. A measure of 1 μg ml−1 of purified collagen VI tetramer was then added to cells. Cells were lysed in IP buffer (0.5% NP-40, 500 mM Tris-HCl pH 7.4, 20 mM EDTA, 10 mM NaF, 30 mM sodium pyrophosphate decahydrate, 2 mM benzamidin, 1 mM PMSF, 1 mM N-ethyl maleimide, cocktail of proteases inhibitors (Roche) and phosphatase inhibitors (Sigma)) 1, 3 and 6 h after collagen VI addition. Protein quantity was measured using BCA assay kit (Pierce) and equal amount of proteins boiled in Laemmli buffer under non-reducing conditions for 5 min before analysis by SDS–PAGE using 4–12% Bis-Tris gradient gels and western blotting with collagen VI antibody and rat monoclonal anti-CMG2 antibody. Actin was used as a loading control.
Cocktail of proteases inhibitors
Manufactured by Roche
The Cocktail of proteases inhibitors is a laboratory product that contains a mixture of chemical compounds designed to inhibit the activity of various proteases, which are enzymes that break down proteins. This product is commonly used in biological and biochemical research to preserve the integrity of protein samples during experimental procedures.
Automatically generated - may contain errors
2 protocols using cocktail of proteases inhibitors
1
Collagen VI Binding and Degradation
2
Quantification of Amyloid Oligomers
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Purified proteins and cellular samples were spotted onto nitrocellulose membrane filters 0.22 μm. The anti-AβOs scFvA13 (3.5 μg ml−1) and scFvIm3 (4.5 μg ml−1) were used as primary antibodies diluted in TBS 0.05% tween, 5% dry milk with the addition of cocktail of proteases inhibitors (Roche). The polyclonal antibody anti-oligomer A11 (ref. 21 (link)) was used 1:500 in TBS 0.05% tween, 5% dry milk. The AβO scFv-positive immunodetection was performed by anti-His tag (for cellular samples), or by anti-V5 tag (for monomers and oligomers of synthetic Aβ, α-Synuclein and lysozyme), which respectively recognizes the C-terminal 6xHis tag or V5 tag of recombinant scFvs (expressed in Escherichia coli and purified, see the paragraph below). Serial dilution curves of cellular samples were preliminarily tested to obtain non-saturating condition of immunodetection. Samples were loaded as follows: 450 ng per dot of synthetic human Aβ1-42; 500 ng per dot of recombinant human α-Synuclein; 500 ng per dot of lysozyme; 20 μl per dot of 15 × concentrated 7PA2 CM and related samples (CM of γ-secretase inhibitor-treated 7PA2 cells and 7PA2-A13K CM) by using Bio-Dot SF apparatus Bio-Rad; 500 ng per dot of total lysates (non-denatured).
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