Novex wedgewell gels

Cortex (≈80–90mg) and hippocampus (≈15mg) isolated from ND and HSD mice were sonicated in 800 and 500μl of RIPA buffer (50mM Tris-HCl pH 8.0, 150mM NaCl, 0.5% Deoxycholic Acid, 0.1% SDS, 1mM EDTA pH 8.0, 1% IGEPAL CA-630, 1mM Na3VO4, 20mM NaF and one tablet/10mL of cOmplete™, EDTA-free Protease Inhibitor Cocktail, Millipore Sigma) and equal volumes were mixed with SDS sample buffer, boiled, and analyzed on 10% or 10–20% Novex™ WedgeWell™ gels (Thermo Fisher Scientific). Proteins were transferred to PVDF membranes (Millipore), blocked at room temperature (RT) for 1 hour with 5% milk in TBS, and incubated, overnight at 4°C, with primary antibodies (see Reporting Summary) in 5% BSA in TBS/0.1% Tween-20 (TBST). Membranes were washed in TBST, incubated with goat anti-mouse or rabbit secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology) for 1 hour at RT and protein bands were visualized with Clarity Western ECL Substrate (Bio Rad) on a Bio Rad ChemiDoc MP Imaging System. Quantification was performed using Image Lab v6.0 (Bio Rad).

After homogenization in cold RIPA buffer and centrifugation, 150μl of the supernatant containing the proteins was boiled at 100°C for 10 minutes. Samples were cooled on ice for 20 minutes and then centrifuged at 20,000 g at 4°C for 15 minutes. The supernatant corresponding to the heat stable (HS) fraction was then harvested. This method is used to isolate proteins resistant to heat including tau and other microtubule-associated proteins. Thus, endogenous immunoglobulins are precipitated during the boiling process and eliminated from the supernatant. The proteins were then mixed with equal volumes of SDS sample buffer, boiled, and analyzed on 10% Novex™ WedgeWell™ gels (Thermo Fisher Scientific). Although tau protein is partially lost during the boiling process, the HS samples are enriched with tau (Extended Data Fig. 5h). Furthermore, boiling significantly improves specificity of certain antibodies such as AT8, RZ3 or MC1³⁸ .

Detection of S-nitrosylated calpain 2 was performed using the biotin-switch technique, as previously described⁴² . Briefly, samples were sonicated in 800μl of RIPA buffer containing 0.1mM of neocuproine and, after centrifugation, protein concentrations were measured. Cysteine thiol groups in 1mg of proteins were blocked with 10% S-methylmethane thiosulfonate (MMTS) (Sigma). After protein-precipitation with 100% acetone, sodium ascorbate was added to the sample to convert each S-nitrothiols (SNO) to a free thiol via a transnitrosation reaction to generate O-nitrosoascorbate. Next, each nascent free thiol (previously an SNO site) was biotinylated with biotin–HPDP (Pierce). Biotinylated proteins were then pull-down by using avidin beads and analyzed on 10% Novex™ WedgeWell™ gels (Thermo Fisher Scientific). Before avidin pulldown, a small fraction of each sample was collected to determine protein “input.” The degree of pulldown correlates with protein S-nitrosylation of calpain 2 or Cdk5 which was detected with an antibody against the two proteins (see Extended Data Suppl. Table 1). Nitrosylation of calpain 2 or Cdk5 is expressed as the ratio between the pull-down signal and the input corrected for the β-actin levels.