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Fig. 2. Characterisation of the antibody that recognises Tau phosphorylated at Thr50. GST-Tau was phosphorylated by 2 U/ml of SAPK3/p38. (A) 0.5 µg of GST-Tau non-phosphorylated (NK) or phosphorylated (SAPK3/p38) was electrophoresed and blotted with the Phos-Thr50 antibody raised against the peptide corresponding to residues 44-55 of Tau phosphorylated at Thr50 or with the Tau antibody that recognises both the unphosphorylated and phosphorylated proteins. (B) Different amounts of phosphorylated GST-Tau or GST-Tau(T50A) were spotted onto a nitrocellulose membrane. Membranes were incubated with 1 µg/ml of Tau antibody, antibody Phos-Thr50 or antibody Phos-Thr181. Immunoblots were carried out in the absence of competing peptide or protein (None), or in the presence of the unphosphorylated peptide immunogen (Thr50 peptide), the phosphopeptide immunogen (Phos-Thr50 peptide), phosphopeptide for another site (Phos-Thr153 peptide, Phos-Ser202 peptide or Phos-Thr69 peptide), a mixture of phosphopeptides (Phos-Thr153 peptide, Phos-Ser202 peptide and Phos-Ser235) or 0.2 mg/ml Phos-Tau(T50A) protein. Peptides were at 100 µg/ml. (C) Phosphorylation of Tau by p38 MAPKs in HEK293 cells. After transfection with plasmids encoding HA-Tau and HA-p38 or HA-p38ß, or myc-SAPK3/p38 or myc-SAPK4/p38, HEK293 cells were exposed for 15 minutes to 0.5 M sorbitol or 0.5 mM sodium arsenite, or for 30 minutes to UV-C (200 J/m²). Five micrograms of cell lysate was immunoblotted using antibody Phos-Thr50 and an antibody that recognises unphosphorylated and phosphorylated Tau equally. To examine activation of p38 MAPKs, 10 µg of cell lysate was used in the immunoblot. The p38 phospho-specific antibody also recognises phosphorylated p38ß, SAPK3/p38 and SAPK4/p38.

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