Fig. 2

SNX3 engages a flippase complex proposed to regulate membrane deformation. a Functional annotation analysis of proteins identified in the SNX3 SILAC proteomics using Gene Ontology annotations with a greater than 3-fold enrichment and with a minimum of two peptides revealed a preponderance of proteins involved in “transport” (47/106), “vesicle mediated transport” (33/106) and “establishment of protein localisation” (33/106). The larger the node the greater the number of proteins classified in that category, while the thicker the edge between nodes the greater the overlap of proteins within those classifications. b Network analysis of putative SNX3 interactome components classified within the “transport” node using the STRING database. c SNX3 does not engage SNX27. HEK293T cells transiently expressing GFP-SNX2 and GFP-SNX3 were immuno-precipitated and analysed for binding to endogenous SNX27. d SNX3 but not SNX1 associates with MON2. Cell extracts derived from RPE1 cells lentivirally transduced with GFP, GFP–SNX3 or GFP–SNX1, were subjected to a GFP nanotrap and subsequently analysed for binding to MON2. e, f DOPEY2 associates with MON2. Cell extracts derived from HEK293T cells transiently transfected with GFP, DOPEY2-GFP or GFP-MON2 were subjected to a GFP nanotrap and subsequently blotted with antibodies raised against endogenous MON2 and DOPEY2. g MON2 can self-associate. Cell extracts derived from HEK293T cells transiently transfected with GFP and MON2-FLAG or GFP–MON2 and MON2-FLAG were subjected to a GFP nanotrap and subsequently blotted with a FLAG antibody. h DOPEY2 can self-associate. Cell extracts derived from HEK293T cells transiently transfected with GFP and DOPEY2-FLAG or DOPEY2-GFP and DOPEY2-FLAG were subjected to a GFP nanotrap and subsequently blotted with an anti-FLAG antibody. i Binding of SNX3 to endogenous MON2 occurs independently of SNX3′s ability to bind to retromer. Cell extracts derived from HEK293T cells transiently transfected with GFP or GFP-tagged wild-type or mutant forms of SNX3 were subjected to a GFP nanotrap and subsequently blotted with an antibody raised against endogenous MON2. C-I are representative blots from three independent biological replicates