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.