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Fig. 4. Knock-down of SAPK3/p38 and SAPK4/p38 inhibits Tau phosphorylation at Thr50 after osmotic stress. (A) Comparison of substrate specificities of different SAPK/p38s. Each kinase (0.2 U/ml) was assayed under initial rate conditions, as described in Materials and Methods. The final concentrations of the Thr50-peptide (black bars) or MBP (grey bars) were 100 µM and 20 µM, respectively. Substrate phosphorylation is plotted as a percentage of the maximal phosphorylation. Results in A are shown as the mean±s.e.m. for triplicate determinations from a single experiment. (B) SH-SY5Y cells were transfected in the absence of siRNA (empty pSuper vector or control GAPDH siRNA), or in the presence of siRNA against SAPK4/p38 (pS1, pS2 and pS3 constructs) or siRNA against SAPK3/p38 (S3mix and pS3mix) as detailed in Materials and Methods. SAPK4/p38 from 10 mg lysate or SAPK3/p38 from 1 mg lysate was immunoprecipitated and immunoblotted as indicated above. (C) Endogenous Tau was immunoprecipitated from the cell lysates indicated in panel B, and immunoblotted as indicated in Fig. 3.

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