Analysis of sarkosyl-insoluble and -soluble tau fractions

Brain cortex tissues from App^NL-G-F and control rats were extracted using either the sarkosyl or the RIPA method. For the sarkosyl extraction, tissues were homogenized in cold RIPA-A buffer without SDS (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM EDTA, pH 8.0, 1% Nonidet-P40, 0.5% Sodium deoxycholate, 50 mM Na-fluoride, 1 tablet of COMPLETE inhibitors and 10 μL 1000 × NaVO₄). The homogenates were centrifuged at 20,000 × g for 20 min at 4 °C, and the supernatants (~2.5 mL) were transferred to 15 mL Falcon tube and add 3.0 mL RIPA-B (RIPA-A buffer with 1% N-lauroylsarcosine (Sarkosyl)). After 1 h incubation with rocking at room temperature, the homogenates were centrifuged for 70 min at 100,000 × g and the supernatants were collected as sarkosyl soluble fractions. To prepare the sarkosyl-insoluble fraction, the pellets were resuspended in RIPA buffer with SDS. After a brief sonication (2× pulses for 10 s at 40% intensity) to dissolve fibrils, the supernatants were collected. Protein concentrations were measured using the BCA kit, and the samples were analyzed with 10% SDS-PAGE gels.