Fig. 6.

The antiangiogenic mβCTPs specifically interact with β3-endonexin. a Lysate aliquots of HUVECs exogenously expressing EGFP-β3-endonexin (EGFP-β3-EN) were incubated with purified GST or GST-fused β CT proteins coupled on Glutathione Sepharose beads. After incubation, the beads were washed and the precipitated proteins were separated by SDS-PAGE. The loaded GST proteins on the beads were assessed by Coomassie blue (C. blue) staining. The co-precipitated EGFP-β3-endonexin and endogenous kindlin-2 (K2) were detected by Western blotting. b Interaction of the β CTs with β3-EN or K2 was evaluated using the yeast 2-hybrid system by a serial dilution method on selection media. Two known interacting molecules (Bop1 and Bop2) were employed here as a positive control, and empty vectors were transformed to serve as a negative control. The growth of yeast cells on SD-2 media (−Leu/−Trp) indicates a successful transformation; the growth on SD-3 selection media (−Leu/−Trp/-His) indicates a positive protein-protein interaction. c The interaction of β3-EN with β3CTP and β5CTP was evaluated using the yeast 2-hybrid system. d Interaction between EGFP-β3-endonexin and GST-β3 CT was evaluated by a pull-down assay in the presence of mβ3CTP or mβ5CTP (20 μM at a final concentration) as described in a