Fig. 1.

SNX27 interacts with Met1 via OTULIN. A SNX27 is selectively enriched by Met1 linkage. Bar plot showed enrichment of SNX27 by di-ubiquitin linkages in mESC and NPC. The raw data was obtained from previous publication [15]. B Streptavidin based immunoprecipitation assay to validate the interaction between SNX27 and di-ubiquitin linkages. Biotin-tagged di-ubiquitin coupled to streptavidin beads were incubated with HeLa whole cell lysate. SNX27 and OTULIN antibodies were used to detect the interaction after immunoblotting. Immunoblotting was performed at least twice, and one representative figure was shown. C Streptavidin based immunoprecipitation assay to validate the interaction between SNX27 and Met1 linkage in HeLa and mES cells. Biotin-tagged K48, K63 and Met1 di-ubiquitin coupled to streptavidin beads were incubated with HeLa or mES whole cell lysate. SNX27 antibody were used to detect the interaction after immunoblotting. Black triangle indicates specific signal. D Interaction proteomics analysis of SNX27. HeLa cells expressing control vector or Flag-SNX27 were lysed and incubated with Flag-M2 beads, after which protein on-bead digestion and peptide desalting were performed before mass spectrometry analysis. Data were analysed using DEP package as described [17]. OTULIN and SNX27 are highlighted. Full dataset is available in Additional file 6: Table S1. E OTULIN mediates the interaction of SNX27 and Met1 linkage in vitro. Bacterial recombinant expressed GST or GST-SNX27 were incubated with Met1 di-ubiquitin in the presence or absence of Flag-OTULIN. Indicated antibodies were used to detect the protein in input and pull-down samples. F OTULIN is required for SNX27 and Met1 linkage interaction in vivo. HeLa cells with inducible shRNA-mediated knockdown for luciferase or OTULIN were used to exam the interaction between SNX27 and Met1 linkage. Cells were treated with doxycycline for indicated time. Immunoprecipitation and immunoblotting assays were performed as B