Fig. 5

Effect of PSEN1 mutations on the interaction between PSEN1 and GSK3 β.

(A)Proteins were immunoprecipitated from equal amounts of cells (±2.10⁶) transiently overexpressing presenilin (PSEN1) using 2.5 µg of monoclonal anti-glycogen synthase kinase-3β (GSK3β) or anti-mouse immunoglobulin G₁ (IgG₁). The immunocomplexes were washed four times in binding buffer, were seperated on a 14% SDS gel and immunoblotted with 1/5000 SB129 (i). 1/720 and 1/360 of the original lysate was evaluated for PSEN1 (iii) and GSK3β (iv) expression, respectively. One-quarter of the immunocomplex was tested for the presence of GSK3β (ii). (B) Proteins were immunoprecipitated from equal amounts of cells (±2.10⁶) stably overexpressing PSEN1 using approximately 10 µg of polyclonal SB129 or anti-rabbit IgG. The immunocomplexes were washed four times in binding buffer, were seperated on a 14% SDS gel, and immunoblotted with 1/4000 anti-GSK3β (i). 1/720 and 1/360 of the original lysate was evaluated for PSEN1 (iii) and GSK3β(iv) expression, respectively. One-quarter of the immunocomplex was tested for the presence of SB129 (ii). colP, co-immunoprecipitation; IP, immunoprecipitation; FL, full length; NTF, N-terminal fragment; WT, wild type; mock.