Fig. 1

Nuclear translocation assay.

(A) Total cell (TC) lysates of stable human embryonic kidney (HEK) 293 cells expressing wild type (wt) or mutant (mt) (I143T, G384A, T113–114ins) PSEN1, were evaluated for presenilin (PSEN1), β-catenin, glycogen synthase kinase-3β (GSK3β), and β-actin expression levels before (0 min) and after (60 min) LiCl stimulation. 10 µg of total proteins were separated on a 10 or 14% Tris/Glycine polyacrylamide gel, blotted onto polyvinylidenedifluoride (PVDF) membranes and developed using the specified antibodies. (B) To obtain cytosolic-free fractions (CFF), cytosolic fractions were incubated overnight with concanavalin A sepharose beads to remove cadherin-bound β-catenin. 5 µg of proteins were separated on a 10% Tris/Glycine polyacrylamide gel and immunoblotting was performed using 1/4000 mouse monoclonal anti-β-catenin. (C) Nuclear free fractions (NFF) were obtained after overnight incubation with concanavalin A sepharose. Immunoblotting was performed using 1/4000 anti-PARP and 1/4000 anti-β-catenin.