Proteasome and deubiquitination activity were inhibited in the most mature (largest) leaves of wild type and amiNAA10 plants by floating leaf discs (4 mm) on ½ × Hoagland medium supplemented with 50 μM of MG132 (Santa-Cruz Biotechnology) and 20 μM deubiquitinase inhibitor PR-619 (Sigma Aldrich) for 5 h. After incubation, samples were frozen in liquid nitrogen. Proteins were extracted in 20 mM sodium phosphate buffer pH 7.2, 1% Tween 20, 10 mM DTT, 0.5 mM PMSF, cOmplete protease inhibitor cocktailTM (Roche), and 20 µM PR-619. The resulting crude extract was clarified by centrifugation (30 min at 4 °C, 15000 g) and incubated with 40 µl beads (Ubi-Qapture Q matrix, Enzo life Sciences) overnight. The proteins were eluted from the matrix according to manufacturer’s instructions. Same volume of the different elution fractions was separated by SDS-PAGE and visualized by silver staining (GE Healthcare). The immunological detection was done with the antibody mono- and polyubiquitinylated conjugated to HRP (FK2, HRP conjugate, Enzo life Sciences) provided with the Ubi-Qapture Q kit.
Silver staining
Manufactured by GE Healthcare
Silver staining is a laboratory technique used to visualize and detect specific proteins or biomolecules in a sample. The core function of silver staining is to amplify the signal of the target molecule, allowing for its detection and analysis.
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Lab products found in correlation
Mg132, by Santa Cruz Biotechnology (1 mentions)
Pr 619, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Ubiqapture q kit, by Enzo Life Sciences (1 mentions)
Protean 2 system, by Bio-Rad (1 mentions)
Multiphor system, by Cytiva (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Incomplete freund s adjuvant, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Nephelometer, by Siemens (1 mentions)
Rabbit anti rps6, by Cell Signaling Technology (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
7 protocols using silver staining
1
Inhibition of Proteasome and Deubiquitination
2
Two-Dimensional Gel Electrophoresis Protocol
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2-DE was carried out using the Multiphor system (Amersham Pharmacia) for IEF and Protean II system (Bio-Rad) for SDS-PAGE. Protein samples (150–200 μg) in 250 μl of solubilization solution (9 M urea, 2% CHAPS, 4 M thiourea, 2% IPG buffer; pH 4–7, 18 mM DDT and bromophenol blue) were loaded onto Immobiline Drystrips (13 cm, pH 4–7) and rehydration was preceded for 12 h at room temperature. IEF was conducted in gradient mode for 1 h at 1000 V, 1 h at 2000 V and 10 h at 8000 V, followed by 8000 V for a total of 65 kVh at 20°C. After the first-dimensional separation, the gel strips were equilibrated for 15 min in equilibration buffer (50 mM Tris-HCl pH 6.8, 6 M urea, 30% glycerol, 2% SDS and bromophenol blue). For the first equilibration, 0.25% DTT was added, and for the second equilibration, 4.5% iodoacetamide was used. SDS-PAGE was carried out in 12% separation gels with constant current of 40 mA/gel. After electrophoresis, the proteins were visualized by silver staining (GE Healthcare) and then the 2-DE images were obtained and analyzed using the Progenesis SameSpots program, v2.0 (Nonlinear Dynamics).
3
Affinity Purification and Western Blot of Fat3
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pCMV-GST-Fat3 constructs were transiently transfected into HEK293 cells, and cells were harvested 72 hours later with a lysis buffer containing 20mM Tris-HCl pH 8.0, 137mM NaCl, 2mM EDTA, 1% NP-40 and 10% Glycerol supplemented with Protease Inhibitor Cocktail (Sigma P8340). Cleared cell lysates were incubated with 50% slurry of Glutathione Sepharose 4B (GE Healthcare Life Sciences) for 2h at 4°C, washed four times with lysis buffer, and eluted by boiling in SDS-PAGE sample buffer. Following polyacrylamide gel electrophoresis on 4–10% gradient gels proteins were visualized by silver staining (GE Healthcare Life Sciences). For Western Blot, proteins samples were resolved by SDS-PAGE, and transferred to nitrocellulose membranes (BioRad) using a transfer buffer containing 25mM Tris and 192mM glycine. For analyzing full length Fat3, transfer buffer was supplemented with 0.05%SDS and 7.5% methanol. Blots were blocked with Tris-buffered saline, 5% non-fat dry milk and 0.05% Tween-20, and incubated with primary antibody (0.5–1ug/ml) overnight, followed by horseradish peroxidase conjugated secondary antibodies (BioRad). Immunoreactive protein bands were detected using ECL 2 western blotting substrate (Pierce).
4
Proteomic Analysis of Extracellular Vesicles
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Proteins were extracted from EV suspensions with 15% (final concentration) of trichloroacetic acid, and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE; 2 µg of protein per well) with 5/12% polyacrylamide gels and run with Tris/glycine/SDS (25 mM/192 mM/1%) buffer. The electrophoresis was performed at 30 mA for 90 min; the proteins were visualized with silver staining (GE Healthcare) and analyzed with ImageJ software 1.50i (National Institutes of Health, Bethesda, MD). Alternatively, for the Western blot analysis, the proteins were transferred to a 0.45 μm pore nitrocellulose membrane (Amersham Biosciences, Little Chalfont, UK) using an electrotransfer tank (0.8 mA/cm2 for 90 min). The membrane was blocked with 5% skimmed milk in PBS for 1 h at 4°C. The membrane was then incubated with 1:7,000 rabbit anti-M. tuberculosis H37Rv soluble extract (MTSE) serum [which was prepared by immunizing a rabbit with 1 mg of MTSE mixed 1:1 with incomplete Freund’s adjuvant (Sigma Aldrich, St. Louis, MO); a total of two immunizations were performed, on day 0 and day 28, and blood was obtained on day 42]. The membrane was left at 4°C overnight. After extensive washing with PBS, peroxidase-labeled anti-rabbit IgG antibody (clone Poly4064, BioLegend), diluted to 1:2,000 was added, and the bands were visualized with 3,3′-diaminobenzidine (Sigma-Aldrich) and H2O2.
5
Visualizing Amyloid-Beta Aggregation Inhibition
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To visualize inhibitory effect of compounds on Aβ aggregation, all samples were prepared under the identical conditions as used in ThT assay. For Aβ cross-linking, 10 mM Ru(Bpy) and 200 mM APS were dissolved in buffer A (0.1 M sodium phosphate, pH 7.4), and diluted with the same buffer to make 1 mM and 20 mM, respectively. Then, 1μL of Ru(Bpy) and APS were added to 10μL of each incubated sample. The mixtures were irradiated by visible light, and the reaction was quenched by adding 3μL of 5X sample buffer containing 5% reducing agent. Then, samples were boiled for 5 min at 95°C and separated by SDS-PAGE electrophoresis. After gel running, different size of Aβ peptides was visualized by silver staining according to the manufacturer’s instructions (GE Healthcare).
6
Multimodal MRI and CSF Analysis
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MRI was performed on 1.5 or 3 Tesla scanners. Diffusion-weighted imaging with calculation of apparent diffusion coefficient map, axial and coronal T1-weighted spin-echo before and after application of gadolinium, coronal and sagittal fluid-attenuated inversion recovery, axial fast T2-weighted field-echo, and axial turbo T2-weighted spin-echo sequences and diffusion tensor imaging were performed in the patients.
CSF cells were counted using a Fuchs–Rosenthal chamber. Total protein levels, integrity of the blood/CSF barrier, and intrathecal Ig synthesis were analyzed using a nephelometer (Siemens) according to the guidelines by the manufacturer. The oligoclonal band pattern was analyzed by electrophoresis and subsequent silver staining (GE Healthcare).
CSF cells were counted using a Fuchs–Rosenthal chamber. Total protein levels, integrity of the blood/CSF barrier, and intrathecal Ig synthesis were analyzed using a nephelometer (Siemens) according to the guidelines by the manufacturer. The oligoclonal band pattern was analyzed by electrophoresis and subsequent silver staining (GE Healthcare).
7
Co-Immunoprecipitation of mRNP Complexes
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Washed IP beads were mixed with 2x LDS sample buffer (Thermo Fisher) containing 50mM DTT (Sigma-Aldrich). Eluted mRNPs from oligo-d(T) 25 precipitates samples as well as input samples were mixed in 1:1 ratio with 4X LDS sample buffer, 100mM DTT. To allow reduction of covalent DSP mediated bonds, the samples were incubated for 20min at 42 C prior to denaturation for 10min at 95 C. To test protein content and presence of candidate proteins by western blotting in inputs and precipitates, samples were resolved by standard SDS-PAGE and visualized by silver staining (GE Healthcare) or blotted onto PVDF membrane (Millipore) and blocked, following instructions in the manufacturer's protocols.
All antibodies utilized for western detection were diluted in PBS, 5% dry milk, 0.1% TWEEN (Thermo Fisher). The primary antibodies (and dilutions) were: rat anti-Y14 (1:2500), rabbit anti-Mago (1:2000, gift of M. Blanchette), rabbit anti-eIF4AIII (1:4,000; gift of I. Palacios), rabbit anti-PABP (1:4000; gift of M. Hentze), rabbit anti-RpS6(1:3000, Cell Signaling) rabbit anti-RpL32 (1:2000; gift of M. Hentze), rabbit anti-Kinesin heavy chain (KHC, 1:25000; Cytoskeleton), rabbit anti-GFP (1:2000, Torrey Pines). Goat anti-rabbit (1:2500) and anti-rat (1:2500) conjugated with HRP (GE Healthcare) were used as secondary antibodies.
All antibodies utilized for western detection were diluted in PBS, 5% dry milk, 0.1% TWEEN (Thermo Fisher). The primary antibodies (and dilutions) were: rat anti-Y14 (1:2500), rabbit anti-Mago (1:2000, gift of M. Blanchette), rabbit anti-eIF4AIII (1:4,000; gift of I. Palacios), rabbit anti-PABP (1:4000; gift of M. Hentze), rabbit anti-RpS6(1:3000, Cell Signaling) rabbit anti-RpL32 (1:2000; gift of M. Hentze), rabbit anti-Kinesin heavy chain (KHC, 1:25000; Cytoskeleton), rabbit anti-GFP (1:2000, Torrey Pines). Goat anti-rabbit (1:2500) and anti-rat (1:2500) conjugated with HRP (GE Healthcare) were used as secondary antibodies.
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