Ahb0042

CA1 whole cell lysates were prepared using tissue from 3-month-old male 5XFAD/PV-Cre mice. Tissue was homogenized in 1 ml RIPA (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) buffer with a hand homogenizer (Sigma), incubated on ice for 15 min, and rotated at 4 °C for 30 min. Cell debris was isolated and discarded by centrifugation at 14,000 rpm for 10 minutes. Lysates were quantitated using a nanodrop and 25 μg protein was loaded on a 10% acrylamide gels. Protein was transferred from acrylamide gels to PVDF membranes (Invitrogen) at 100 V for 120 min. Membranes were blocked using bovine serum albumin (5% w/v) diluted in TBS:Tween. Membranes were incubated in primary antibodies overnight at 4 °C and secondary antibodies at room temperature for 90 minutes. Primary antibodies were anti-APP (Invitrogen; PAD CT695), anti-APP (Sigma;A8967), anti-β-actin (Abcam; ab9485). Secondary antibodies were horseradish peroxidase-linked (GE Healthcare). Signal intensities were quantified using ImageJ 1.46a and normalized to values of β-actin. We examined tau protein solubility using sequential protein extraction as described in Yoshiyama et al., 2007.^{28 (link)} We then probed the detergent insoluble tau fraction using an antibody against Tau5 (Thermo Fisher Scientific; AHB0042).

Immunoblot was performed as previously reported [26 (link)], with slight modification. Precast 4–20% tis-glycine or 10–20% tris-tricine gels were used (Bio-Rad). The iBlot^™ Gel transfer device (Invitrogen) was used for transfer. The membranes were incubated in blocking buffer (Blocking Buffer for Fluorescent Western Blotting Rockland MB-070) for 1 h at room temperature. The following primary antibodies were used: 6E10 (Covance SIG-39300, 1:1000), APP, C-terminal (Sigma A8717, 1:1000), actin (Sigma A2066, 1:1000), actin (Sigma A3853, 1:1000), phospho-tau Ser404 (Thermo Fisher Scientific #44-758G, 1:2000), phospho-tau pThr181 (Thermo Fisher Scientific #701530, 1:1000), tau (Thermo Fisher Scientific #AHB0042, 1:2000). Antibodies were diluted in the blocking buffer, and membranes were incubated overnight at 4°C. The membranes were washed three times with TBST and secondary antibodies (Odyssey IRDye 680 or 800, all 1:10000) were applied for 1 h at room temperature. After washing four times, proteins were visualized using a Licor Odyssey Infrared imaging system. Blots were analyzed using ImageJ 1.47v software (National Institutes of Health). For the detection of Aβ, the membranes were microwaved in PBS to unmask the epitopes and increase the immunoblot sensitivity before blocking [69 (link)].

Hippocampus and auditory cortex from 6-month-old tau P301S mice were dissected and homogenized in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitors. Lysates were incubated on ice for 15 min and spun at 12,000 RPM for 15 minutes. Then, supernatants were transferred to fresh tubes and analyzed for protein concentration (Bio-Rad Protein Assay). Equal amounts of protein (20 ug/lane) was resolved on a SDS-polyacrylamide gel and blotted onto a PVDF membrane. This membrane was incubated in blocking buffer containting 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% (v/v) Tween 20 (TBS-T) plus 5% dry milk (m/v) for 1 h at room temperature followed by incubation overnight at 4 °C in primary antibodies and then secondary antibodies at room temperature for 1 hour. Primary antibodies were anti-phospho-tau (Ser396) and anti-phospho-tau (Thr181). Secondary antibodies were LI-COR IRDye secondary antibodies. Signal intensities were quantified using ImageJ 1.46a and normalized to values of total tau Tau5 (Thermo Fisher Scientific; AHB0042).