Bis tris criterion gels

Solubilized membranes from WT and REST KO MEFs were incubated with 2 μM C100-FLAG substrate in HEPES buffer at pH 7.0 with 0.1% phosphatidylcholine, 0.025% phosphatidylethanolamine, 0.00625% cholesterol, and a final CHAPSO concentration of 0.25% for 1 h at 37 ^°C. Equal amounts of solubilized protein were added to each reaction based on the protein concentration determined by BCA assay. AICD-FLAG product was detected by incubating equal volumes of WT and REST KO reactions in SDS sample buffer for 10 min at room temperature. The reaction samples were run on 12% Bis Tris Criterion gels (Bio-Rad), followed by transfer of proteins to PVDF, western blotting with M2 anti-FLAG antibody (Sigma), and exposure of the blot using ECL (GE Healthcare). AICD bands were then quantified by densitometry using ImageQuant software (GE Healthcare).

Crushed frozen tissue was homogenized in RIPA buffer (R0278, Sigma-Aldrich, St. Louis, MO, USA), supplemented with 0.1% protease (cOmplete, Roche, Basel, CH) and phosphatase inhibitor (PhosStop, Roche, Basel, CH), and equalized to a protein content of 1.8 μg/μl. Following denaturation and reduction, homogenized lung samples (15 μl) were loaded onto 4–12% Bis-Tris Criterion gels (3450125, Bio-Rad, Veenendaal, NL). After the transfer of proteins to nitrocellulose (162-0115, Bio-Rad, Veenendaal, NL), membranes were blocked with 5% milk (ELK, Friesland Campina, Amersfoort, NL). Blots were incubated overnight with the primary antibodies (details described in Supplementary Table 2) at 4 °C, followed by incubation with the secondary antibody (details described in Supplementary Table 2). Protein bands were visualized with Pico Chemiluminescent Substrate kit (34580, Thermo Fisher, Breda, NL) and intensities quantified using ImageQuant TL (v8.1.0.0). Protein contents of β-actin were used to normalize the data and presented as fold changes compared to controls.

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).

Cells cultured in 6-well plates were lysed on ice with a buffer containing 25 mmol/l Tris/HCl pH=6.8, 3 mmol/l EDTA, 3 mmol/l EGTA, 50 mmol/l NaF, 2 mmol/l sodium orthovanadate, 270 mmol/l sucrose, 10 mmol/l b-glycerophosphate, 5 mmol/l sodium pyrophosphate, and 0.5% Triton X-100 and protease and phosphatase inhibitor cocktails (Sigma). Protein concentration was measured using the BCA Assay (Thermo Scientific, Paisley, UK), lysates were diluted with sample loading buffer (Life Technologies, Paisley, UK) and 20 μg of protein was loaded per well and separated on gradient 4–20% Bis-Tris Criterion gels (Bio-Rad, Hemel Hempstead, UK), transferred onto nitrocellulose membranes using the iBlot Dry Transfer System (Life Technologies), incubated with a blocking solution of 5% BSA in 0.05% TBST buffer and incubator overnight with primary antibodies (antibodies are listed in Supplementary Table S4) in 0.05% TBST, followed by 30 min washing in 0.05% TBST, and incubation with HRP-tagged secondary antibodies (Cell Signalling Technologies, New England Biolabs, Hitchin, UK) for 1 h and subsequent washing in 0.05% TBST for 30 min. Luminescence was detected with Syngene ChemiGenius using Super-Signal West Dura Chemiluminescence Substrate (Thermo Scientific).