Supplementary figures:

Suppl. fig.1. Deglycosylation of mature legumain. Purified bovine legumain (1 µM) was

incompletely deglycosylated by incubation with different PNGase F dilutions for 10 minutes at 37oC.

Suppl. fig.2. HCT116 cell viability is not influenced by tunicamycin. HCT116 cells were treated

with 2.5 or 5 µg/ml tunicamycin for 24 hours and presented relative to control (DMSO-treated)( n=3).

Suppl. fig.3. Purified mature bovine legumain is internalized. HEK293 cells (5*104 cells) were

cultured on sterile cover slips and incubated for 24 hours in media with (lower panel) or without (upper

panel) purified bovine legumain (5 µM). Legumain (green) and nuclei (blue) were visualized using

fluorescence microscopy. Merged panels, right. Scale bars represent 10 µm.

Suppl. fig.4. Purified bovine legumain alone or complexed with the activity-based probe MP-L01

were internalized. HEK293 cells (5*104 cells/well; 48-well plate) were incubated for 24 hours in media

containing DMSO (-), purified bovine legumain alone (10 µM), probe alone (0.5, 2.5 and 50 µM) or a

complex of legumain prebound to the probe (0.5-10 µM). Legumain (red; upper panel and second panel),

probe (green; upper panel and third panel) and merged panels (upper panel) were detected by

immunoblotting. GAPDH was used as loading control (lower panel).