Figure 7.

Internalization of PN-1, plasmin, and plasmin-PN-1 complexes in vSMCs. PN-1 alone (125 nM, A,F), plasmin alone (25 nM, B), and pre-formed plasmin-PN-1 complexes (C,D,E,G) were added to vSMCs for 2 h at 37°C without (A–E) or with RAP (50 μg/ml, F,G). After cell permeabilization, PN-1 alone (A,F) was detected by Alexa Fluor®488-labeled secondary antibody (green), plasmin alone (B) by Alexa Fluor®555-labeled secondary antibody (red) and nuclei by DAPI staining (blue). Plasmin-PN-1 complexes were revealed with plasmin antibody and detected by Alexa Fluor®555-labeled secondary antibody (C,G, red) or PN-1 and plasmin antibodies and detected with Alexa Fluor®488 (D, green) and 555-labeled secondary antibody (D, red) respectively. Yellow color and white arrows highlight examples of intracellular co-localization of plasmin-PN-1 complexes revealed with PN-1 and plasmin antibodies (D). LRP-1 was detected with Alexa Fluor®555-labeled secondary antibody (E, red), plasmin-PN-1 complexes were revealed with plasmin antibody and detected by Alexa fluor®488-labeled secondary antibody (E, green). Yellow color and white arrows indicate the intracellular colocalization of LRP-1 and plasmin-PN-1 complexes (E).