Fig. 3. Motif-dependent activities of IL-10RB and IL-2Rγ.

a Motif-containing peptides in IL-10RB and IL-2Rγ. Critical residues highlighted in red font were mutated to alanine in order to explore motif-dependent functions. IL-2Rγ Y303 is not strictly part of the motif, but it was also mutated since Y favors binding to the WDD at all positions of the peptide. b Co-precipitation studies between wild-type or motif-mutated versions of the receptors and GST-WDD. HEK-293T cells were co-transfected with Flag-tagged wild-type (WT) or motif-mutated (5 M or 6 M) forms of the indicated tagged cytokine receptors and a construct expressing GST-WDD (320–607). 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. c Evaluation of motif-dependent LC3 lipidation activity. HEK-293T cells were transfected with the indicated versions of HA-tagged IL-10RB and IL-2Rγ (WT or 5 M) along with a plasmid expressing AU-LC3. Cells were lysed 36 h post-transfection for Western-blotting against the indicated molecules. d Evaluation of motif-dependent co-localization between the cytokine receptors and GFP-ATG16L1. HeLa cells were transfected with the indicated versions of Flag-tagged IL-10RB and IL-2Rγ (WT or 5 M) and a plasmid expressing GFP-ATG16L1. Cells were fixed 36 h post-transfection, stained with an anti-Flag antibody and scored for colocalization between the Flag (red) signal and GFP-ATG16L1. Shown are representative confocal pictures (left, where the white arrows indicate colocalization events), and graphs (right) representing the percentage of Flag-positive vesicles colocalizing with the GFP-positive signal −/+ s.d. (left axis, white bars, solid dots) together with the total number of Flag-positive vesicles per cell −/+ s.d. (right axis, gray bars, white dots); n = 20 cells for IL-10RB and n = 23 cells for IL-2Rγ; n.s: P > 0,05; **P < 0.01; ****P < 0.0001, two-sided Student’s t-test.