Figure 2

CLEC14A binds MMRN2 between residues 530 and 624. (a) Diagrams of MMRN2 truncation mutants, showing the elastin microfibril interface (EMI) domain, coiled-coil (CC) domain and complement related C1q domain. The minimal CLEC14A-binding fragment is highlighted in black. (b and c) HEK293T were transfected with truncation mutants, lysates were separated by SDS–PAGE under reducing conditions and far western blotted with CLEC14A-ECD-Fc and western blotted with His tag antibodies. (b) The smallest binding fragment was identified as MMRN2^487–820, MMRN2^FL fails to bind CLEC14A-ECD-Fc. (c) Further truncation mutants were far western blotted revealing the smallest MMRN2 fragment binding CLEC14A is MMRN2^530–624. (d) Purified MMRN2^495–674 and MMRN2^495–603 bind to streptavidin under reducing conditions after biotinylation. (e) Flow cytometry histograms of HEK293T transfected with GFP-tagged CTLD group 14 family members and stained with biotinylated MMRN2^495–674 (grey line), confirming binding to CLEC14A and CD93 but not thrombomodulin. Streptavidin-PE alone was used as a control for background binding (grey shaded).