Figure 4

Cystatin E/M over-expression in melanoma cells suppressed legumain activity: A375 and MCC11 cells were transfected with pCST6 (closed bars) or empty pTracer vector (control; open bars), media were changed the following day, and serum-free conditioned media and cell lysates were collected 24 h later. (A) Immunoblots of cystatin E/M in conditioned media (CM) and lysates. Secreted proteins were TCA-precipitated prior to SDS-PAGE and immunoblotting. The filters were stained with a cystatin E/M-specific (upper and middle panels) or a β-actin specific (lower panel; loading control) antibody, respectively. (B) Inhibitory activity against legumain was measured in the conditioned media as residual legumain activity using fluorometry, a fixed amount of partially purified legumain from rat kidney and the substrate Z-Ala-Ala-Asn-NHMec (A375, p < 0.0001; MCC11, p = 0.002). (C) Activity of legumain in cell lysates measured by cleavage of Z-Ala-Ala-Asn-NHMec (A375, p < 0.0001; MCC11, p = 0.0012). Error bars represent the standard error of the mean (SEM).