Figure 7

Co-immunoprecipitation of APP, VPS35 and SorLA in COS-7 and HEK293 cells. (A) Endogenous APP and VPS35 were co-immunoprecipitated in COS-7 cells using the anti-APP N-terminus 22C11 and the anti-VPS35 antibodies (αVPS35). Negative controls (C) were performed by immunoprecipitating cells with the same secondary antibodies, sepharose- (IP VPS35 control) and agarose- (IP 22C11) linked, respectively. (B) Transfected Wt APP-GFP and endogenous VPS35 co-immunoprecipitate from COS-7 cells using the indicated antibodies (Ab). (C) Transfected Wt, S655A and S655E APP-GFP were immunoprecipitated in COS-7 cells with the anti-GFP antibody, and the co-immunoprecipitated endogenous VPS35 forms were detected with an anti-VPS35 antibody. Asterisk (*), non-specific IgGs bands (IgGs raised in goat) of the IP samples. Non-transfected cells (C(nt)) submitted to the same IP procedures (incubated with primary and agarose-linked secondary antibodies) were used as control in (B) and (C). (D) HEK293T cells were cotransfected with SorLA cDNA, APP₆₉₅ and eGFP (transfection control). Immunoprecipitation was performed using antibodies raised against the C-terminus of APP (369), the N-terminus of SorLA (αSorLA) or using pre-immune serum as negative controls (C). Immunoprecipitation and co-immunoprecipitation were detected by western blot (WB) using the anti-APP 369 antibody and an anti-SorLA C-terminus antibody (αSorLA C-term). Immunoblot analysis included GFP as an additional negative control.