Mab1510

To assess polyubiquitination levels in the UBI4 repeat variants during HS, overnight-saturated cultures were diluted in 50 ml YPD and grown at 30 °C till OD₆₀₀ = 0.6–0.8. Before HS, we harvested cells from 10 ml cultures and transferred the remaining cultures to a water bath at 44 °C. At 15, 30, and 60 min after temperature switch, we again harvested cells from 10 ml cultures. Cell pellets were lysed with glass beads in cold lysis buffer (50 mM Tris (pH 7.4), 100 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, and protease inhibitor cocktail (Roche)) in a FastPrep-24 homogenizer (MP Biomedicals). Extracts were cleared by centrifugation (10 min, 14,000 r.p.m. at 4 °C) and protein concentrations were measured by the Bradford assay (Protein Quantification kit, Sigma-Aldrich). Total proteins (5 μg) were separated by 10–20% gradient gels (Life Technologies) using a Tricine-based buffer, followed by transfer to polyvinylidene difluoride membranes. Blots were incubated with mouse anti-ubiquitin monoclonal antibody MAB1510 (Millipore, 1:2000) followed by incubation with rabbit anti-mouse IgG-horseradish peroxidase-conjugated antibody (ab97046, Abcam, 1:20,000), and western signals were detected using the LumiGLO Ultra Chemiluminescence kit (KPL).

For visualization of EGFP-tagged constructs, cells grown on glass coverslips were fixed with 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for 10 min, then washed with PBS and counterstained with DAPI Nucleic Acid Stain (Invitrogen). For ubiquitin labeling, N2a cells were fixed in methanol for 10 min at −20°C, re-hydrated with PBS for 10 min and blocked for 30 min in 5% (w/v) bovine serum albumin with 0.3% (w/v) Triton X-100 in PBS at room temperature. Immunocytochemistry was performed using monoclonal antibody recognizing ubiquitin (mAb1510; Chemicon) at 1:1000 diluted in PBS. Antibody distribution was visualized by epifluorescence microscopy after incubation with secondary antibody conjugated to Alexa 594 diluted at 1:350 in PBS (Molecular Probes), and finally counterstained with DAPI Nucleic Acid Stain. Labeled cells were visualized using a Leica DM6000 microscope and digital images captured using Volocity imaging software (PerkinElmer).

Dual immunofluorescence was performed on spinal cord tissue from two SALS cases using a rabbit polyclonal anti-TDP-43 antibody (Proteintech, 10782-2-AP) and mouse monoclonal anti-ubiquitin antibody (Chemicon, MAB1510). Five μm spinal cord sections were deparaffinised with xylene, and rehydrated with a descending series of diluted ethanol and water. Antigens were retrieved by heating sections in 10 mM citrate buffer (pH 6.0). Non-specific background was blocked with 1% bovine serum albumin (BSA) (Sigma Aldrich). Sections were incubated at 4 °C overnight with the primary antibodies (1:1000 for anti-TDP-43 and 1:100 anti-ubiquitin), followed by incubation with secondary antibodies (anti-rabbit conjugated with Alexa Fluor 488 and secondary anti-mouse conjugated with Alexa Fluor 555; Life Technologies) for 1 hour at room temperature. Slides were coverslipped using the Prolong Gold antifade reagent.
Confocal fluorescence imaging was performed using a Leica DM6000 upright laser scanning confocal microscope with Leica application suite advanced fluorescence software. Images were acquired with a 40 × oil immersion objective. Images were acquired using sequential mode to avoid crosstalk between two dyes.

Seven μm thick paraffin-embedded block sections of frontal cortex, anterior striatum, and hippocampus (CA1 sector) from sCJD MM(V)1 (n = 5), VV2 (n = 5), MV2K (n = 5) brains lacking any AD- or Lewy body (LB)-related co-pathology and controls lacking significant neurodegenerative pathology (n = 2) were stained with hematoxylin-eosin and processed for immunohistochemistry with a monoclonal anti-ubiquitin antibody with proven specificity [29 (link),30 (link)] (MAB1510, dilution 1:1000, Chemicon, Tomecula, CA, USA) as described [27 (link),30 (link)]. A mean combined score was given to each case as a result of two operators semi-quantitative assessments of ubiquitin immunopositivity (0, no immunoreactivity; 1, mild; 2, moderate; 3, prominent immunoreactivity).

Human skin fibroblasts cultures were treated with epoxomicin (15 nM) or pre-treated with trehalose (100 mM) 15 min before the treatment with epoxomicin for 24 h. Cells were washed with PBS plus phenylmethylsulfonyl fluoride (PMSF) (SIGMA-Aldrich, P7626), scraped in 150 μL of lysis buffer [50 mM Tris HCl, 150 mM NaCl, 20 mM EDTA, 1% Triton X-100, 50 mM sodium fluoride (NaF), 20 mM N-ethyl-maleimide, 100 μM sodium ortovanadate, 1 mM PMSF and protease inhibitors cocktail (Calbiochem, 539131)] and immediately boiled for 5 min. The lysates were centrifuged at 12.000×g at 4°C for 30 min. The supernatant was used for protein determination by the BCA protein assay kit. For detection of ubiquitinated proteins by western blot, 15 μg of protein were conducted to immunoblot assay with a mouse monoclonal antibody to ubiquitin diluted 1/500 (Chemicon, MAB1510). The secondary antibodies (1/1000) followed by ECL (enhanced chemiluminescence) detection reagents (Bio-Rad, 970442/3) were used for immunodetection. Immunoblot of β-actin diluted (1/5000) (SIGMA-Aldrich, A5441) was performed to demonstrate equal protein loading. The blots were quantified by computer-assisted video.