Fig 1: Clever-1 harbors an interaction motif for the multiprotein vacuolar ATPase complex. A, Representative confocal images of FP-1305 and 9-11 internalization into primary human macrophages after 2-hour antibody treatment (N = 2 healthy donors). A single focal plane and orthogonal view of the indicated magnified area shows vesicles containing either one or both of the antibodies. Colocalization was assessed with Manders colocalization coefficients M1 (FP-1305) and M2 (9-11); N = 10 cells from two independent experiments with the same donor. Scale bar, 5 µm. Statistical comparison of co-localization was conducted using Wilcoxon matched-pairs signed rank test. B, Schematic of the main 9-11 and FP-1305 antibody epitopes mapped on the human Clever-1 protein primary structure. FAS1, fasciclin domain; LAM, laminin-type EGF-like domain; LINK, C-type lectin-like hyaluronan-binding LINK module. C, Venn diagram showing the number of 9-11– and FP-1305–specific proteins and proteins shared by both antibodies in the CRAPome-pruned Clever-1 interactome. D, High-confidence protein–protein interactions in the Clever-1 interactome retrieved from the STRING database and mapped using Cytoscape. Proteins present in the 9-11 interactome have a green border and proteins present in the FP-1305 interactome a yellow center. The five largest clusters are highlighted with grey boxes and titled after common themes in their significantly enriched GO Biological Process terms. E, Significantly enriched pathways from IPA performed separately on 9-11–specific, FP-1305–specific, and shared proteins. F, Magnification of the Clever-1 interactome showing subunits of the v-ATPase complex, which immunoprecipitated specifically with 9-11. G, Coimmunoprecipitation/Western blot validation showing that the v-ATPase subunits ATP6V1A, ATP6V0A1, and TCIRG1 are immunoprecipitated with 9-11 but not with FP-1305. Clever-1 signal was detected with the 3-372 antibody, which is the parent antibody of FP-1305. Rat IgG2a and human IgG4 served as isotype controls in coimmunoprecipitation for 9-11 and FP-1305, respectively. **, P < 0.01.