Fig. 2
Tau phosphorylation precludes the BIN1-Tau interaction in vitro. a. Tau-F5 fragment phosphorylation analysis after in vitro phosphorylation by CDK2/CycA3 kinase. Representative immunoblots using various antibodies against phosphorylation epitopes in Tau. The total amount of Tau-F5 was revealed by the phosphorylation-independent antibody Tau5. The significant shift in molecular weight observed in CDK2/CycA3-treated samples indicates Tau hyperphosphorylation. In contrast to other antibodies, the Tau-1 antibody binds to various non-phosphorylated Tau sites; the signal thus decreases when Tau is hyperphosphorylated [31]. b. 2D [1H, 15N] HSQC spectra of 125 μM 15N CDK-phosphorylated Tau-F5[165–245] free in solution (gray) and with a 1 molar amount of GST-BIN1/SH3 (red, superimposed): Overlaid details of 2D [1H, 15N] HSQC spectra presented in Additional file 6. c. 2D [1H, 15N] HSQC spectra of 60 μM 15N CDK-phosphorylated 2N4R Tau free in solution (gray) and with a 2 molar amount of GST-BIN1/SH3 (blue, superimposed): Overlaid details of full spectra presented in Additional file 7