Figure 3: LYVE-1 is a substrate of MT1-MMP.

(a) Western blotting analyses of MT1-MMP expression in LECs and BMMs from Mmp14^f/f Tie1-Cre⁻ and Mmp14^f/f Tie1-Cre⁺ mice. (b) Morphometric comparison of corneal lymphatic vascularization in Mmp14^f/f Tie1-Cre⁻ and Mmp14^f/f Tie1-Cre⁺ mice (left panel). Quantification of vascularized area was shown in the right panel (**P<0.01, n=5, two-tailed t-test). Data represent the mean±s.e.m. (c) MT1-MMP sheds LYVE-1 in primary LECs. The conditioned media and total cell lysates from WT and Mmp14^−/− LECs were examined by western blotting analyses using antibodies indicated. Data are representative of three independent experiments. (d) HEK293T cells expressing C-terminally Flag-tagged LYVE-1 were transfected with either WT or E/A catalytic mutant MT1-MMP (MT1 EA). The conditioned media and total cell lysates were subjected to western blotting analyses using antibodies indicated. Data are representative of three independent experiments. (e) rLYVE-1 was incubated with recombinant catalytic domain of MT1-MMP at two enzyme/substrate ratios (1:50 [+], 1:10 [++] and buffer only [−]). The protein mixture was subjected to western blotting analyses using specific antibody indicated. The cleaved fragments of LYVE-1 are indicated by black arrows. Data are representative of three independent experiments. (f) A diagram illustrating the predicted cleavage sites of LYVE-1 by MT1-MMP. (g) Endogenous interaction between LYVE-1 and MT1-MMP. LYVE-1 and MT1-MMP immunoprecipitations (IP) were generated from WT LECs using specific antibodies against LYVE-1 and MT1-MMP, and examined by western blotting analyses using indicated antibodies. IgG immunoprecipitation was used as controls. The experiments were repeated at least three times.