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Fig. 3. Phosphorylation of endogenous Tau in SH-SY5Y human neuroblastoma cells. (A) SH-SY5Y cells were incubated for 6 hours in medium with (1) or without (2) serum and 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). Endogenous Tau was immunoprecipitated with an anti-Tau antibody from 1.0 mg of cell lysate and immunoblotted using the Phos-Thr50 antibody or an antibody that recognises both unphosphorylated and phosphorylated Tau. To examine SAPK/p38 activation, 50 µg of cell lysate was used in the immunoblot with the p38
phospho-specific antibody, as shown in Fig. 2. (B) SH-SY5Y cells were preincubated for 1 hour with or without 10 µM SB203580 or 10 µM PD184352 prior to a 15 minutes exposure to 0.5 M sorbitol. Tau protein was immunoprecipitated and immunoblotted as indicated in A. Endogenous SAPK4/p38
was immunoprecipitated from 10 mg cell lysate with anti-SAPK4/p38 antibody, and immunoblotted using the p38 phospho-specific antibody, or with an antibody that recognises both unphosphorylated and phosphorylated SAPK4/p38. Activation of other p38s was examined as described in A. 60 µg of cell lysates were immunoblotted with an antibody that recognises MAPKAP-K2 phosphorylated at Thr334 or with an antibody that recognises both unphosphorylated and phosphorylated MAPKAP-K2.
