His₆-tag allows internalization of EDIN in endothelial cells through PA₆₃.

<p>A: Upper panel: SDS-PAGE of recombinant His₆-tagged EDIN before (left) and after thrombin treatment (right). Lower panel: immunoblot anti–His₆-tag on His₆-tagged EDIN before and after cleavage by thrombin. B, C: Immunoblots showing cellular levels of active RhoA (RhoA-GTP) in HUVECs determined by GST-Rhotekin RBD pulldown (labeled RhoA-GTP). Cellular content of RhoA (Total RhoA) was assessed by anti-RhoA on 2% of total protein extracts. Immunoblot anti-actin antibody exhibits equal protein loading. (B) Cells were intoxicated with different concentrations of His₆-EDIN (1, 10 and 100 µg/ml) with and without 3 µg/ml of PA₆₃, as indicated. (C) Cells were intoxicated with 10 µg/ml His₆-EDIN, 10 µg/ml EDIN, and 3 µg/ml PA₆₃ as indicated.