Western Blot Analysis of Protein Markers

Cells were lysed in RIPA buffer (MilliporeSigma) supplemented with proteinase (ThermoFisher) and phosphatase (PhosSTOP, Roche Diagnostics, Indianapolis, IN) inhibitors. The concentration of cell lysates was determined using bicinchoninic acid assay (BCA) (ThermoFisher). Samples were loaded onto 4–20% Mini-Protean TGX Precast Gels (Bio-Rad) and then transferred to PVDF membranes (Bio-Rad) using Trans-Blot Turbo System (Bio-Rad). Membranes were incubated with following primary antibodies: mouse anti-total tau A0024 (DAKO; 1:1000, # A0024), mouse anti-synapsin (ThermoFisher; 1:1000, MA531919), rabbit anti-Gapdh-HRP (Cell Signaling; 1:2000, #8884), rabbit anti-Rgp antibody CAB101 (1:1000) [1 (link)], and rabbit anti-Kgp antibody CAB102 (1:1000) [1 (link)]. All secondary antibodies were horseradish protein (HRP) conjugated (Vector; 1:5000 or 1:10000). Blots were imaged by chemiluminescent detection using Supersignal West Fento (ThermoFisher) and the ChemiDoc imaging system. Quantification was performed with Image Lab 6.0.1 software (Bio-Rad) by dividing the integrated OD of all bands of interest by the OD of GAPDH detected on the same blot.