3.7. Tau Protein Phosphorylation Assay

COS-7 cells transfected with pDSRed2-humanTau (2N4R isoform) and cultured in DMEM/5% fetal calf serum for 24 h in 12-well culture plates were incubated with the respective compounds each 8 µM or 0.1% DMSO as vehicle control for 12 h at 37 °C. Cells were scraped, centrifuged for 10 min at 4 °C with 300× g, washed with cooled PBS and centrifuged again. Proteins were extracted with 300 µL PBS, 1% Triton X-100 including protease and phosphatase inhibitors (cOmplete^TM, PhosSTOP^TM, Roche Life Science, (Germany, Penzberg). Protein content was calculated by the Pierce 660 nm protein assay (Thermo Scientific, Rockford, IL, USA). Ten micrograms of protein from each sample were run as duplicates on 8% SDS-PAGE and transferred to a PVDF membrane. Total tau protein was labelled with a polyclonal total tau antibody (1:4000, v:v, #A0024, Dako, Glostrup, Denmark) and phospho-tau (pThr231, pSer235) was labelled with the monoclonal antibody AT180 (1:500, v:v, MN1040, Thermo Scientific). Western blots were imaged after incubation with anti-rabbit HRP/anti-mouse HRP (1:10,000, v:v NA934, NA931, GE Healthcare, Little Chalfont, UK) using the DNR Chembis ECL station and immunoreactive bands were quantified by densitometry using Tina 2.09 software (Raytest, Straubenhardt, Germany). Phospho-tau immunoreactivity was normalized to total tau immunoreactivity.