Figure 2.

Identification of the potential CD146 dimer interface

(A) Superposition of CD146 D4-D5 on the corresponding domains of the ICAM-1 D3-D5 dimeric form. CD146 D4 is shown in light pink, whereas D5 is shown in hot pink. One monomer of ICAM-1 D3-D5 is shown in yellow, whereas the other is shown in light blue. The two residues, K398 in CD146 D4 and K439 in CD146 D5, that contribute to the dimerization in the chemical cross-linking test shown at the left are drawn in stick representation. Also shown in stick representation are residues that are crucial to the CD146 D4 dimerization, including L392 and L394 in CD146 D4 and W441 in D5. The sodium dodecyl sulphate-polyacylamide gel electrophoresis (SDS-PAGE) results of chemical crosslinking are shown in the inset (left). Lane before: CD146 D4-D5 protein, Lane after: CD146 D4-D5 mixed with chemical cross-linking reagent BS³ and incubated on ice overnight. A covalently linked CD146 D4-D5 dimer appears in the BS³-treated sample.

(B) Enlargement of the part enclosed with dashed lines in A, showing the residues involved in crosslinking, which are crucial for the D4 dimerization.

(C and D) Western blot analysis of the expression of different mutant constructs in the 293T cell line (C) and in the HUVECs (D).

(E) Cartoon schematic of the double-tag co-immunoprecipitation procedure to identify the existence of CD146 dimer on cell surface.

(F and G) Double-tag co-immunoprecipitation assay analysis of the dimerization of CD146 in the 293T cell line (F) and HUVEC cell line (G). The samples in C and D were run on nonreducing SDS-PAGE, and the samples in F and G were run on reducing SDS-PAGE.

See also Figure S4.