Figure 1. Endogenous PS1 and GSK3β exhibit similar expression profiles during neuronal development, partially colocalize in the same vesicular compartment, and coimmunoprecipitate from brain homogenates. A, Western blot analysis of MBO and soluble fractions during development of hippocampal neurons in culture at 1, 3, and 10 d in vitro (DIV). The figure shows samples of three different homogenates obtained at each time point. Note that the expression pattern is similar for PS1 and GSK3β. Both proteins are highly enriched in the MBO fraction, particularly at 10 DIV. PS1 FL, Full-length PS1; PS1 CTF, C-terminus fragment of PS1. The antibodies include anti-GSK3β (334–348), 1:1000, and anti-PS1 (AD3L), 1:2000. B, PS1 and GSK3β colocalize on a subpopulation of MBOs and are enriched in a growth cone fraction. The Western blots show the localization of PS1, GSK3β, and kinesin-I heavy (KHC) and light (KLC) chains in three vesicle fractions (V0, V1, and V2) and supernatant (Cyt). Significant amounts of PS1 and GSK3β colocalize in V1 where kinesin-I is enriched. PS1 and GSK3β are highly enriched in growth cone fractions (GCs). PS1 NTF, N-terminus fragment of PS1. The antibodies include anti-PS1 (PSN2), 1:1000; anti-KHC (H2), 1:2000; anti-KLC (63–90), 1:2000; and anti-KLC ALL, 1:2000. C, Coimmunoprecipitation of PS1 and GSK3β from Cos cell lysates cotransfected with PS1 and GSK3β expression vectors (Cos Lys) and mouse brain homogenates (Brain Lys). A sample of transfected Cos cell lysates was included as a positive control for GSK3β staining (Lys). The Cos cell lysate that was immunoprecipitated with anti-PS1 antibody (Anti-PS1) shows immunoreactivity with anti-GSK3β in the Western blot. Similarly, anti-PS1 immunoprecipitate from mouse brain homogenate shows immunoreactivity for GSK3β. No GSK3β immunoreactivity is observed in immunoprecipitates for nonimmune mouse IgG (Ctrl IgG) or protein A-Sepharose beads alone (Beads). Immunoprecipitations were performed with anti-PS1 antibody PSN2. Western blot was revealed with anti-GSK3β (Calbiochem).