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. 2017 Dec 11;25(5):918–934. doi: 10.1038/s41418-017-0023-1

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© ADMC Associazione Differenziamento e Morte Cellulare 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — RAB37 interacts with ATG5 in a GTP-dependent manner. a A schematic representation of the RAB37 mutants. Inactive RAB37-T43N is a GDP-stabilised mutant, and active RAB37-Q89L is a GTP-stabilised mutant. b, c Protein quantification was determined by Pierce BCA protein assay kit. Three RAB37 proteins (wild type and two mutants) were purified from E. coli. d, e RAB37 (and its mutants -Q89L and -T43N) GTPase activity assays by Quantichrome ATPase/GTPase assay kit according to a standard curve. f RAB37 interacts with ATG5 in a GTP-dependent manner. MYC-ATG5 was transiently co-transfected with FLAG-RAB37-WT, FLAG-RAB37-T43N, FLAG-RAB37-Q89L or control plasmid into 293T cells respectively. For co-immunoprecipitation, the lysates were immunoprecipitated with an anti-FLAG antibody followed by immunoblotting with an anti-MYC and anti-FLAG antibodies. GAPDH was used as an internal control. g RAB37 transfection promotes formation of ATG5 positive puncta in a GTP-dependent manner. COS-7 cells transfected with CHERRY-RAB37-WT, CHERRY-RAB37-Q89L, CHERRY-RAB37-T43N or control vector pSico-CHERRY respectively. Left: representative images of cells with ATG5-GFP (green) and CHERRY-RAB37 (its mutants or control vector CHERRY, red). Right: ATG5 positive dots were quantified from ~20 cells. Data are presented as means ± S.D. * stands for P < 0.05, ** stands for P < 0.01 (n = 3 independent experiments)