Figure 4
CD248 binds to MMRN2 in a distinct region from CLEC14A and CD93 binding. (a) HEK293T transfected with GFP, CLEC14A-GFP, CD248-GFP, and chimeras CLEC14ACD248(sushi) and CLEC14ACD248(CTLD) were lysed and far western blotted with MMRN2FL and western blotted with anti-GFP. MMRN2FL binds to CLEC14A and both chimeras CLEC14ACD248(sushi) and CLEC14ACD248(CTLD) but not CD248-GFP or GFP alone. (b) Immunoprecipitations of GFP-tagged proteins after cell surface biotinylation. CLEC14A, THBD, CD93 and all four CLEC14A chimeras bind to streptavidin showing they are cell surface expressed. GFP alone was included to demonstrate intracellular proteins are not cell surface biotinylated. (c) MMRN2 truncation mutants were transfected into HEK293T and lysates under reducing conditions were far western blotted with mCD248-ECD-Fc, revealing binding to minimal fragment MMRN2133–486. His tag western blot confirmed expression of each MMRN2 protein fragment. (d) mCD248-ECD-Fc pull-downs from HUVEC lysates resulted in enrichment of MMRN2 compared with hFc control. (e) Immunofluorescence analysis of HUVEC stained with MMRN2 mouse polyclonal antibodies, hFc, mCD248-ECD-Fc or CLEC14A-ECD-Fc. MMRN2 antibody staining partially co-localizes with CD248-ECD-Fc and CLEC14A-ECD-Fc binding; scale bar, 40 μm. (f) ELISAs of mCD248-ECD-Fc bound to plate capturing MMRN2 FL His, (**P<0.01 Mann–Whitney test, n=5) error bars represent s.e.m. (g) ELISAs of mCD248-ECD-Fc capturing MMRN2 FL His and then binding by CLEC14A-ECD-Fc detected by anti-CLEC14A antibody C2. (**P<0.01 Mann–Whitney test, n=5) error bars, s.e.m. (h) Diagram of CLEC14A or CD93 expressed by endothelial cells binding to MMRN2 in the ECM, which in turn is bound by CD248 expressed by fibroblasts or vasculature associated pericytes. CD248, CLEC14A and CD93 binding is due to the CTLD, CD248 binds MMRN2 in the region 133–486, whereas CLEC14A binds in the region 530–624.
