Figure 1.

Co-immunoprecipitation (IP) of the Nef-ATG8 complex. Co-IP studies were carried out with lysates prepared from HEK293 cells stably expressing Nef-DsRed (A–C) and cotransfected with plasmids encoding for the indicated YFP-ATG8 constructs (D–F). (A) and (D) show samples before immunoprecipitation. In (A) an anti-GABARAPL2 (a), an anti-GABARAP (b), an anti-GABARAPL1 (c), and an anti-LC3B antibody (d) were used to detect the different ATG8s. (B) Immunoprecipitates with anti-DsRed antibody followed by SDS-PAGE and immunoblotting with various ATG8 antibodies (a: anti-GABARAPL2, b: anti-GABARAP, c: anti-GABARAPL1 and d: anti-LC3B). (C) In a reciprocal set of experiments, immunoprecipitates were carried out with various ATG8 (a: anti-GABARAPL2, b: anti-GABARAP, c: anti-GABARAPL1 and d: anti-LC3B) antibodies followed by SDS-PAGE and immunoblotting with an anti-DsRed antibody. In (D) an anti-DsRed antibody was used to detect Nef fused to DsRed and an anti-YFP antibody to detect YFP and the ATG8s fused to YFP. (E) Immunoprecipitates with an anti-DsRed antibody or (F) with an anti-GFP antibody followed by SDS-PAGE and immunoblotting with the antibodies are indicated. Data are representative of three independent experiments (U: unbound material; E: eluate fraction). Full-length western blots are presented in Supplementary Figs 1 and 2.