Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy Schematic presentation of ATG16L1 fragments and mutants.

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Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy Schematic presentation of ATG16L1 fragments and mutants used in this study. The following fragments encompassed the indicated amino acids: wild type (WT): 1–623; ∆1: 39–632; ∆2: 120–623; ∆3: 206–623 and ∆4: 336–623. Mutant ∆2∆182–205 consists of the ∆2 fragment with an additional deletion in amino acids 182–205. All constructs contained a Flag‐S tag at the N‐terminal end. Fragments depicted in (A) were expressed in U2OS cells and amino acid starved for 5 h followed by fixation and immunostaining against S tag to detect S‐ATG16L1. Scale bar: 10 μm. U2OS cells expressing Flag‐ATG16L1∆2 or Flag‐ATG16L1∆2∆182–205 were treated as in (B) and stained using antibodies against Flag tag (to detect ATG16L1, green) and FIP200 (red). Scale bar: 10 μm. Protein–protein interaction assay in 293T cells transiently transfected with the indicated Flag‐S‐tagged ATG16L1 constructs. S tag pull‐down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies. Homodimerisation assay in 293T cells transiently transfected with ATG16L1‐GFP and the indicated Flag‐S‐tagged ATG16L1 constructs. S tag pull‐down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies. Homodimerisation assay similar to (E). Flag‐ATG16L1WT or Flag‐ATG16L1∆182–205 were stably expressed in ATG5−/− cells and analysed by immunofluorescence using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO J, Volume: 38, Issue: 9, First published: 01 April 2019, DOI: (10.15252/embj.2018100554)