Cells were lysed in 1% NP40 lysis buffer with protease inhibitor cocktail (Roche). Equal protein amounts were loaded
in LDS sample buffer (NuPAGE, ThermoFisher) onto 4%−12% gradient SDS-PAGE gels, and run with NuPAGE antioxidant
(ThermoFisher). After blotting onto 0.2 μm pore PVDF membranes (Merck), nonspecific background was blocked in 5% w/v
skimmed milk (Tesco) or 10% BSA in PBS containing 0.1% Tween-20 for 1 h and incubated with primary antibody overnight at
4°C, followed by the appropriate HRP-labeled secondary antibody. Blots were developed with ECL Dura (GE Healthcare).
For the dot blot, FACS-sorted cells were pelleted and lysed in lysis buffer. DNA was digested by the addition of 50 mg/ml
DNase-I (Sigma) in 50 mM TrispH 8 with 10 mM MgCl2 for 1 h at 37° C. Samples were incubated in 5% SDS in
dH2O for 10 min at room temperature and the entire mixture was filtered through a cellulose acetate membrane
under vacuum. Membranes were washed in 5% SDS under vacuum, blocked in 5% skimmed milk in PBS for 30 min, then incubated with
primary antibody (HT7) overnight at 4°C, and developed using the method described above. Asarkosyl-insoluble tau
preparation from a 5 m P301S-tau mouse brain (Allen et al., 2002 (link)) and was used as a
positive control.
in LDS sample buffer (NuPAGE, ThermoFisher) onto 4%−12% gradient SDS-PAGE gels, and run with NuPAGE antioxidant
(ThermoFisher). After blotting onto 0.2 μm pore PVDF membranes (Merck), nonspecific background was blocked in 5% w/v
skimmed milk (Tesco) or 10% BSA in PBS containing 0.1% Tween-20 for 1 h and incubated with primary antibody overnight at
4°C, followed by the appropriate HRP-labeled secondary antibody. Blots were developed with ECL Dura (GE Healthcare).
For the dot blot, FACS-sorted cells were pelleted and lysed in lysis buffer. DNA was digested by the addition of 50 mg/ml
DNase-I (Sigma) in 50 mM TrispH 8 with 10 mM MgCl2 for 1 h at 37° C. Samples were incubated in 5% SDS in
dH2O for 10 min at room temperature and the entire mixture was filtered through a cellulose acetate membrane
under vacuum. Membranes were washed in 5% SDS under vacuum, blocked in 5% skimmed milk in PBS for 30 min, then incubated with
primary antibody (HT7) overnight at 4°C, and developed using the method described above. Asarkosyl-insoluble tau
preparation from a 5 m P301S-tau mouse brain (Allen et al., 2002 (link)) and was used as a
positive control.