Fig. 2. Selection of candidate receptors showing WDD-dependent functional features.

a Co-immunoprecipitation studies to identify receptors able to specifically interact with the WDD. HEK-293T cells were co-transfected with tagged forms of the indicated cytokine receptors along with constructs including the WDD (320–607) or ΔWDD (1–319) fused to GST, as shown. Cells were lysed 36 h later and subjected to GST immunoprecipitation with agarose beads coupled to GSH. Shown are Western-blots against the indicated molecules. IP: immunoprecipitate; TL: total lysate. b Evaluation of WDD-dependent LC3 lipidation activity. ATG16L1−/− HCT116 cells restored with the indicated versions of ATG16L1 (full-length (FL) or separated N-terminal (Nt; 1–299) and C-terminal (Ct; 300–607) domains) were transfected with the shown tagged cytokine receptors along with plasmids expressing AU-LC3 and GFP. Cells were lysed 36 h post-transfection for Western-blotting against the indicated molecules. The expression levels of co-transfected GFP remain unchanged, indicating equal transfection efficiency. c Colocalization between transfected cytokine receptors and co-expressed GFP-LC3. HeLa cells were transfected with the indicated tagged cytokine receptors and a plasmid expressing GFP-LC3. Cells were treated with bafilomycin A1 (75 nM, as indicated) during the last 6 h of culture, fixed 36 h post-transfection, stained with an anti-Flag antibody and scored for colocalization between the Flag signal (red) and GFP-LC3. Shown are representative confocal pictures (left) where the white arrows indicate colocalization events. Graphs (right) display the percentage of Flag-positive vesicles colocalizing with the GFP-positive signal in the different conditions −/+ s.d. (n = 23 cells for IL-10RB; n = 25 cells for IL-2Rγ; n.s: P > 0,05; ***P < 0.001, two-sided Student’s t-test).