Fig.2.

Localization and interaction of Pyk2 and tau. A) Representative immunofluorescence staining of Pyk2 in primary hippocampal neurons (DIV (days in vitro) 10) cultured in microfluidic chambers. Cells were counterstained for tau (all compartments including axons) and MAP2 (all compartments excluding axons). DAPI labels the nuclei. Scale bar: 100μm. B) Co-immunoprecipitation of Pyk2 and tau in RAB fractions of brain lysates obtained from WT and pR5 mice reveals weak interaction of endogenous Pyk2 and tau in vivo. Note that cleaved forms of endogenous Pyk2 were detected in the input lysate. IP, immunoprecipitation. Three independent experiments were performed. C) Endogenous Pyk2 is present in the crude synaptosomal fraction, together with tau. WT brain tissue was processed to obtain fractions including total homogenate (Hm), supernatant S1, pellet P1 and pellet P2, followed by immunoblotting with the indicated antibodies. D) Pyk2-V5-transfected hippocampal neurons (DIV16) stained with Pyk2 and V5, revealing strong localization of Pyk2 in dendritic spines. Scale bar: 50μm. E) WT hippocampal neurons (DIV16) stained with PSD95 (postsynaptic marker) and MAP2. Colocalization of endogenous Pyk2 and PSD95 shown in spines of a WT neuron indicated by arrowheads. Scale bar: 50μm.