Figure 3.

AA98 blocks the dimerization of CD146 to depress EC activation

(A) CD146 D4-D5 in complex with AA98 Fab. CD146 is shown in ribbon representation, D4 is colored in light pink, whereas D5 is colored in hot pink. AA98 Fab is shown in the surface module, and the light and heavy chains are colored in pale cyan and light blue, respectively. Residues L392, L394, and W441, which are crucial for CD146 dimerization, are shown in stick representation.

(B) The activation of NF-κB signaling caused by the expression of different mutant constructs of CD146 was analyzed by Western blot.

(C) HUVECs transfected with different mutant constructs of CD146 were subjected to a transwell migration assay. Data were represented as mean ± standard deviation. Significant differences were determined by one-way analysis of variance (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, no significant difference). Representative images are shown in Figure S7A. Similar expression level of CD146 wild type and mutants was confirmed by Western blot (inset, top).

(D) Co-immunoprecipitation assay analysis of mutant constructs Δ387E-399R/K439C, Δ387E-399R, and W441A and of wild-type CD146 expressed in the 293T cell line with AA98.

(E) Western blot analysis of the dimerization of CD146 in mutant-transfected 293T cells treated with AA98 or not.

(F) Transwell assay analysis of the migration activity of mutants transfected with HUVECs treated with AA98 or not.

Data were represented as mean ± standard deviation. Significant differences were determined by one-way analysis of variance (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, no significant difference). Representative images were shown in Figure S7B. Similar expression level of CD146 wild type and mutants was confirmed by Western blot (inset, top).

See also Figures S5–S7.