Functional characterization of recombinants rCXCL4 and rCXCL4L1 in vitro. (A) Alignment of CXCL4, CXCL4L1 and CXCL4-241 amino acid sequences. The fully conserved (.) and substituted residues were indicated. α-helix (gray shade) of CXCL4 is represented. The alignment was constructed using ClustalW. (B) In vitro endothelial cell migration assay using the scratch assay. BAECs were stimulated with FGF2 (10 ng/mL) in the presence or absence of rCXCL4L1 (0.02 and 0.05 μg/mL) or rCXCL4 (1 and 5 μg/mL). (C) Quantification of the scratch assay results (n = 6). (D) In vitro endothelial cell migration assay using Boyden chambers. In comparison, the effect of CXCL4L1 or CXCL4 on endothelial cell migration was also tested on HUVECs (n = 6). (E-F) In vitro endothelial cell proliferation using MTT assay. BAECs and HUVECs were stimulated with FGF2 (10 ng/mL) in presence or absence of various concentrations of rCXCL4 or rCXCL4L1 and cell proliferation was determined.