Proteomic quantification of the SNX-BAR interactome identifies the IGF1R as a SNX-BAR interactor. (A) GFP-tagged SNX1, SNX2, SNX5, and SNX6 were transiently expressed in SILAC-labeled HEK293 cells. GFP-only was expressed in nonlabeled HEK293 cells as a control. The SNX-BARs and GFP were precipitated with GFP-trap beads, each SNX-BAR IP was pooled with a GFP-only IP, and the combined samples were quantified by LC-MS/MS. The panel displays the fold enrichment of the other SNX-BARs in each individual SNX-BAR precipitation, indicating robust heterodimerization and multimerization between the SNX-BARs. Selected other interactors of the respective SNX-BAR protein at least fivefold enriched over the GFP-only control are indicated by arrows. (B) GFP-trap IPs of the indicated SNX-BARs and analysis of the presence of the endogenous IGF1R and the INSR in the precipitates. (C) GFP-trap IPs of the isolated cytoplasmic tails of selected potential SNX-BAR cargoes and analysis of the presence of the endogenous SNX-BAR protein in the precipitates. IB, immunoblot. (D) Direct recombinant interaction with the GST-tagged cytosolic tails produced in bacteria and His-tagged SNX5, and SNX6 coexpressed with His-tagged SNX1 and SNX2, respectively. (E) Immunofluorescent staining of endogenous IGF1R (green) and endogenous SNX1 (red) in serum-starved MCF-7 cells treated with 10 nM IGF-1 for the indicated periods. (F) Degradation assay of endogenous IGF1R and the INSR in HeLa cells, and clonal SNX1/2 and SNX5/6 double-KO cells lines treated with the ribosomal inhibitor cycloheximide for the indicated periods. The degradation kinetics were quantified over four independent experiments. Bars, 10 µm. Error bars indicate SD. *, P < 0.05 compared with control conditions in a t test.