0.45 μm nitrocellulose membrane

Cells were washed with PBS, lysed in RIPA buffer (50 mm Tris/HCl pH 7.4; 1% NP‐40; 0.5% Na‐deoxycholate; 0.1% SDS; 150 mm NaCl, 2 nm EDTA, 50 mm NaF) containing complete protease inhibitor cocktail (Roche, F.Hoffmann‐La Roche Ltd, Basel, Switzerland) for 30 min on ice, and cleared by centrifugation. Protein concentration was determined using Pierce BCA assay kit (Thermo Fisher Scientific Inc.) with a BSA standard curve. Before loading, protein lysates were denatured at 95 °C for 5 min in 6× SDS sample buffer. Proteins were separated by SDS/PAGE on 7.5 or 10% gels before semi‐dry transfer to 0.45 μm nitrocellulose membranes (GE Healthcare, Chalfont St Giles, UK) and blocked in 5% dry milk powder in TBS‐T (100 mm Tris, pH 7.5, 0.9% NaCl, 0.05% Tween‐20). Membranes were incubated with primary antibodies diluted in 5% BSA in TBS‐T at 4 °C over night. After washing in TBS‐T, near‐infrared labeled secondary antibodies (IRDye, Li‐Cor Biosciences, Lincoln, NE, USA, dilution 1 : 5000) were applied for 4 h at room temperature. Images were acquired using Azure c600 fluorescent imager.
Cells for protein extraction for western blots shown in Figs 4C and S6 were synchronized in M phase using 5 μm S‐Trityl‐L‐cysteine (Sigma‐Aldrich) and harvested by mitotic shake off before lysis in RIPA buffer.

Cells were lysed in boiling buffer (10% SDS, 0.5 M Tris–HCl pH 6.8) and centrifuged at 20,000×g for 30 minutes; 20 μg of protein were separated on 7.5-15% SDS-PAGE and transferred onto 0.45-μm nitrocellulose membranes (GE Healthcare Europe, Milan, Italy). Blots were stained using standard procedures and signals were revealed by a chemiluminescence reagent (Euroclone, Milan, Italy). Horseradish peroxidase (HRP)-linked secondary antibodies, anti-phospho-Ataxia Telengectasia (Ser 1981) (ATM), anti-ATM, anti-phospho-p53, anti-p53 and anti-vinculin were from Cell Signaling Technology (Beverly, USA); anti-phospho-Histone H2A.x (Ser 139) was from Millipore (Temecula, CA) and anti-Histone H2A.x was from GeneTex (San Antonio, Texas). Three independent experiments were performed.

10 to 20 female heads, thoraces or abdomens were homogenized by sonication in 100 μL of ice-cold RIPA buffer supplemented with Complete mini without EDTA protease inhibitor (Roche). Protein concentration was measured using the BCA protein assay kit (Pierce) according to the manufacturer’s instructions. 10 μg of total proteins were supplemented with 2× LDS containing reducing agent (Invitrogen) and heated at 100 °C for 10 minutes prior to loading on 10% Bis-Tris Criterion gels (Biorad). Proteins were transferred to 0.45 μm nitrocellulose membranes (GE Healthcare) that were subsequently blocked in TNT buffer (Tris–HCl 15 mM pH 8, NaCl 140 mM, 0.05% Tween) with 5% non-fat dry milk for 1 h at room temperature and incubated overnight at 4 °C with antibodies recognizing either full-length dTau (1/10,000) or β-Actin (1/200,000, Abcam). HRP-conjugated anti-mouse or anti-rabbit antibodies (1/10,000, Invitrogen) were used for 1 h at room temperature and detection was performed using an ECL chemiluminescence kit (GE Healthcare) and Hyperfilms (GE Healthcare).

Equal protein quantities and volumes of all samples were loaded onto 10% NuPAGE Bis-Tris gels (invitrogen), and transferred onto 0.45 μm nitrocellulose membranes (GE Healthcare). Membranes were blocked during 1 h at room temperature in PBS 1X containing 0.1% Igepal and 3% BSA.
Membranes were analysed using the following antibodies: anti-HA rat monoclonal antibody (Roche, 1:2,500); anti-Nsr1p mouse monoclonal antibody (Abcam, 1:5,000), anti-NCL rabbit polyclonal antibody (Abcam, 1:5,000), anti-GAPDH (Sigma, 1:5,000), anti-EBNA1 mouse monoclonal antibody (OT1X, 1:2,000), anti-OVA rabbit polyclonal antibody (Sigma, 1:2,500), anti-actin (Sigma, 1/5,000). The membranes were then washed with fresh PBS 1X+0.1% Igepal and incubated for 45 min with swine anti-rabbit or goat anti-mouse secondary antibodies (Dako) conjugated to horseradish peroxidase at a 1:3,000 dilution, and analysed by enhanced chemiluminescence (ECL, GE Healthcare) using a Vilbert-Lourmat Photodocumentation Chemistart 5000 imager. All the experiments were repeated at least three times. Relative protein levels for each sample were normalized to GAPDH or Actin protein levels as indicated, using Fusion-Capt Advance software.

For Western blot analysis under reducing conditions, 2 μg of each Aβ peptide was loaded per lane after heating at 95°C for 5 minutes on 4 - 12% Tris-Tricin VarioGels (Anamed), transferred to 0.45 μm nitrocellulose membranes (GE Healthcare) and detected using the primary antibodies AB5-3 (diluted 1:500) and 4G8 (diluted 1:2000). Running and transfer buffers were applied according to the manufacturer. The blots were developed using enhanced chemiluminescence according to the manufacturer (Roth).