Tau FKBP-FRB-Rapamycin Interaction Assay

The tau FKBP-FRB-rapamycin assay was performed as described previously [53 (link)]. HEK293 cells were transfected with 25 ng of FKBP-tau and FRB-tau, incubated at 37 °C for 4 h, washed with phosphate-buffered saline (PBS), treated with or without 400 nM rapamycin (Calbiochem) for 1 h, washed with PBS and fixed in 4% PFA for tau-PLA experiments. Images of fixed cells were obtained using the DV Elite system based on an Olympus IX71 fully motorized widefield deconvolution inverted microscope with a 60× objective 1.40 numerical aperture (NA) fitted with a CoolSNAP HQ2 cooled charge-coupled device (CCD) camera (Photometrics) driven by SoftWoRx 5.0 software (Applied Precision). Several cell positions were chosen randomly and recorded using the motorized stage. z-stacks were acquired at 0.250 µm intervals to cover the entire volume of each cell. Quantification of tau-PLA puncta was performed on deconvolved images using Fiji [55 (link)]: tau-PLA signals were enhanced using a median filter and then separated from the background using Otsu’s method of thresholding. Signals were detected and counted.