Fig. 3

Gigaxonin promotes the ubiquitination of ATG16L1. a In vivo ubiquitination assay for ATG16L1. COS-7 cells were transfected with Ch-ATG16 alone, or in combination with His-Ubiquitin construct (His-Ub) in the presence or absence of short interfering RNA (siRNA) against endogenous Gigaxonin (Gig siRNA) or the mismatch counterpart (Gig ms) and treated with MG132. The pull down of ubiquitinated ATG16L1 is performed in denaturing conditions using nickel agarose beads. b Colocalisation of ubiquitin with ATG16L1 upon Gigaxonin expression. COS-7 cells were transfected with Flag-Gig, Ch-ATG16 and HA-Ubiquitin (Ha-Ub) constructs ± MG132. The expression of Gigaxonin induces a drastic degradation of Ch-ATG16, and all remaining ATG16L1 (as revealed by +) colocalises with Gigaxonin and ubiquitin. c Gigaxonin promotes the K48-ubiquitination of ATG16L1. Specific labelling of K48 ubiquitin chain did not show substantial colocalisation of this ubiquitin motif on ch-ATG16, when transfected alone in COS-7 cells. In presence of Flag-Gig, Ch-ATG16L1 was degraded and fully decorated by K48-ubiquitin, as shown by the colocalisation of anti-K48 antibodies with ch-ATG16 in boosted pictures ± MG132. d Additional evidence for the ubiquitination of ATG16L1 by the Gigaxonin-E3 ligase using proximity ligation assay (PLA). The physical proximity between ubiquitin and ATG16L1 was revealed after transfection of HA-Ubiquitin with Cherry-ATG16L1 ± Flag-Gigaxonin under MG132 treatment. The specificity of the reactions of the PLA assay is provided in Supplementary Fig. 1a. e The poly-ubiquitination of the endogenous ATG16L1 is promoted by Gigaxonin. PLA was performed on COS-7 cells, transfected only with the HA-Ub ± Flag-Gigaxonin under MG132 treatment. The specificity of the reactions of the assay is provided in Supplementary Fig. 1b. Left panel: PLA amplification using anti-HA/anti-ATG16L1 antibodies. Right panel: The PLA amplification using anti-polyubiquitin chains/anti-ATG16L1 antibodies. Scale bar: 10 µm and 30 µm for zoom pictures