Fig. 5. Effects of Tau mutations T50E and T50A on tubulin polymerisation and reassembly of microtubules. (A) Polymerisation of tubulin promoted by GST-Tau(WT), GST-Tau(T50A) and GST-Tau(T50E) was monitored over time by measuring turbidity. A typical experiment is shown; similar results were obtained using at least three separate preparations of each protein. (B) HEK293 cells transfected with Tau(WT), Tau (T50E) or Tau(T50A) were treated with nocodazole for 2 hours. The microtubule network in transfected cells is shown 20 minutes after nocodazole had been washed out. Immunofluorescence staining was performed using a monoclonal antibody that recognises ß-tubulin. The percentages of cells with re-polymerising microtubules were determined by counting five different optical fields containing at least 200 cells per field. ^** indicates that the differences between the three groups were statistically significant (P<0.01). (C) HEK293 cells transfected with Tau(WT), Tau(T50A) or Tau(T50E) were exposed to 0.5 M sorbitol for 15 minutes. Five micrograms of cell lysate was immunoblotted using antibody AT270, which recognises phospho-Thr181 in Tau, and an antibody that recognises unphosphorylated and phosphorylated Tau.