Figure 2. The expression, purification and binding analysis of FITC-labeled disintegrins by flow cytometry.

Panel A – Coomassie stained gel showing the migration of Trx-VCN before and after TEV proteolysis (lanes 2 and 3, respectively) versus C18 reverse phase-HPLC purified VCN (lane 4). The same amount (5μl) of cell lysates before and after TEV proteolysis was loaded on a precast gel and Coomassie stained. Panel B – VCN and native CN exhibit an almost identical dose-dependent inhibitory effect against ADP-induced platelet aggregation when incubated with human platelet-rich plasma (with a calculated IC₅₀ of ~60nM). In contrast, a recombinant construct based on the exact CN sequence (designated rCN), which is also expressed as a soluble polypeptide in Origami B (DE3), shows no inhibitory activity. Panel C – Labeled disintegrins (FITC-CN and FITC-VCN) bind in a similar manner to cells displaying different integrin profiles as determined by flow cytometry. MDA-MB-435, MDA-MB-231 or HUVEC were either incubated with FITC-CN (green) or FITC-VCN (red) or probed with an FITC-labeled irrelevant antibody control (blue).