Figure 4

Characterisation of a link between BIN1 and Tau. (a) Representative confocal images showing the subcellular distribution of BIN1 and Tau. SKNSH-SY5Y cells were transiently transfected with BIN1 (iso.1) and Tau (1N4R). After 24 h, cells were fixed in paraformaldehyde and staining with anti-BIN1 (green) (99D) and anti-Tau (red) (Tau c-Ter) antibodies. (b) Representative confocal images showing the intracellular distribution of BIN1 and Tau. SKNSH-SY5Y cells were transiently transfected as in a and pre-permeabilized with 0.01% of saponin before fixation to remove an important part of cytosol. Arrows denote colocalization staining between BIN1 and Tau. (c) SKNSH-SY5Y cells were transiently transfected with Tau (1N4R) and BIN1 (iso.1) or control empty plasmid (control). After 24 h, cells extractswere immunoprecipitated (IP) with anti-BIN1 (99D) or anti-Tau (Tau-5) antibody. Precipitated proteins were resolved by SDS-PAGE and visualized with anti-BIN1 (99D) and anti-Tau (Tau c-Ter) antibodies using True-Blot system. (d) GST-Tagged protein or GST alone was incubated with lysate from HEK293 cells overexpressing Tau (1N4R) or BIN1 (iso.1). Alternatively, GST-BIN1 was incubated with 6His-Tau purified protein. Pull-downs were resolved by SDS-polyacrylamide gel electrophoresis and visualized with Coomassie or by western blot using anti-Tau (Tau c-Ter) and anti-BIN1 (99D) antibodies. (e) GST-Amph or GST alone was incubated with lysate from HEK293 cells overexpressing Tau (1N4R). Pull-downs were resolved by SDS-polyacrylamide gel electrophoresis and visualized with Coomassie or by western blot using anti-Tau (Tau c-Ter) antibody. (f) Synaptosomes fraction were extracted from mouse brain. Precleared lysates were incubated for immunoprecipitation with anti-Tau (mTau-5), anti-BIN1 (99D) antibody or with protein G-coupled beads alone (control). Pull-downs were resolved by SDS-polyacrylamide gel electrophoresis and visualized by western blot using True-Blot system.

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