Figure 3

MMRN2 binds to CLEC14A in the C-type lectin domain. (a) CLEC14A-ECD-Fc pull-downs were pre-incubated with either PBS, mouse control IgG, or CLEC14A monoclonal antibodies C1–5 and used to pull down MMRN2^FL from HEK293T-transfected lysates. C1, C4 and C5 blocked MMRN2 enrichment, whereas C2 and C3 did not. (b) Flow cytometry analysis of CLEC14A-ECD-Fc binding to HUVEC surface. CLEC14A-ECD-Fc was pre-incubated with the same blocking conditions as in pull-downs. C1, C4 and C5 significantly blocked CLEC14A-ECD-Fc binding to HUVEC surface. (*P<0.05 Mann–Whitney test n=4) error bars represent standard error mean (s.e.m.). (c) HEK293T transfected with CLEC14A domain deletions were lysed and far western blotted with MMRN2^FL under non-reduced conditions. Upon detection of the MMRN2^FL His tag, MMRN2 binding was observed in all mutants except those lacking the CTLD or sushi domains. Probing with anti-CLEC14A antibodies was included to show expression of each mutant protein. (d) Flow cytometry analysis of HEK293T transfected with CLEC14A wild-type (wt) with a GFP tag, chimera 1 CLEC14A^THBD(CTLD) or chimera 2 CLEC14A^THBD(sushi). All of the CLEC14A monoclonal antibodies and MMRN2^495–674 bound to CLEC14A wt. None of the CLEC14A monoclonal antibodies nor the MMRN2^495–674 fragment bound to chimera 1 CLEC14A^THBD(CTLD) except modest binding with C2. All antibodies except C2 bound to chimera 2 CLEC14A^THBD(sushi). The MMRN2^495–674 fragment could also bind chimera 2 CLEC14A^THBD(sushi).