Figure 1.
Establishment of HEK293-tau-BiFC cell line.
(a) N- and C-terminal constituents of Venus protein was fused to full-length tau (441 a.a.). (b) Basal fluorescence intensity of tau-GFP and tau-BiFC cell line. Scale bar = 200 μm. (c) Expression and basal phosphorylation levels of tau-GFP and tau-BiFC cell line. Immunoblot with anti-tau (ser 262) antibody indicates the expression levels of total tau and immunoblot with anti-tau phospho (ser 396) antibody indicates the basal level of phosphorylated tau. Anti-actin indicates loading controls.
Figure 2.
Cellular distributions of HEK293-tau-GFP (a) and HEK293-tau-BiFC (b).
Cells were incubated with nocodazole or vinblastine (3 μM) for 30 min and fluorescence images were taken. Scale bar = 10 μm.
Figure 3.
Maturation of tau-BiFC upon tau phosphorylation.
(a) Diagram of BiFC maturation upon tau phosphorylation. (b) Tau-BiFC cells were incubated with okadaic acid (30 nM) and forskolin (20 μM) for 24 hrs. Scale bar = 200 μm. (c) Quantification of BiFC-fluorescence increase at various time points. (d-f) For the immunoblot assay, tau-BiFC cells were incubated with compounds for 24 hrs and cell lysates were prepared. Black arrows indicate full-lenth tau, red arrows indicate tau dimers, and blue arrows indicate tau fragments. (g-h) The relative amount of phosphorylated tau including its cleaved forms was normalized with that of non-phosphorylated tau (TauSer262). Error bars indicate s.d. from two independent experiments.
Figure 4.
Cellular distribution of tau-BiFC fluorescence.
(a) HEK293-tau-GFP control (b) HEK293-tau-BiFC cells were incubated with each compound for 24 hrs.