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. 2009 Apr 30;4(4):e5412. doi: 10.1371/journal.pone.0005412

Figure 6. BBK32 (153–175) aggregates plasma Fn.

Figure 6

(a) BBK32 (21–205), BBK32 (146–166), BBK32 (153–175), and BBK32 (175–202) were titrated into 1 µM purified plasma Fn and the optical density at 600 nm was measured. (b) BBK32 (153–175) is the minimal amino acid sequence required to affect Fn matrices assembled by NHDFs. Cells were allowed to grow for 48 hours and then were incubated with 5 µM BBK32 (153–166), BBK32 (146–166), BBK32 (153–166), and BBK32 (175–202). Treated cells were incubated for 20 hours and probed with Alexa Fluor 488-anti-Fn and monoclonal antibody IST 9. Images were taken using LSM 510 Confocal Microscope, objective 63X/1.4 oil. Bar = 10 µm. Staining with monoclonal antibody IST 9 indicates that only the BBK32 peptide containing residues 153–175 changes the conformation of Fn matrices. (c) BBK32 (153–175) is the minimal amino acid sequence required to inhibit HUVEC proliferation. Cells were seeded onto tissue culture plates in full media and allowed to adhere for two hours. After two hours, BBK32 (21–205), BBK32 (153–175), BBK32 (175–202), and anastellin were added in increasing concentrations (in triplicate) to the wells. Cells were then incubated for 72 hours at 37°C, 5% CO2. After 72 hours, cells were washed, trypsinized, and counted (three counts/well, three wells/dose) using a hemacytometer.