Figure 3
From: The aspartic protease napsin A suppresses tumor growth independent of its catalytic activity
Functional analysis of wild-type and mutated napsin A proteins. (a) Recombinant SP-BΔC was synthesized in the baculovirus system, and napsin A and its mutant were expressed in HEK293 cells. Recombinant SP-BΔC was incubated with napsin A or its mutant at pH 4.7 at 37°C for 2 h. Napsin A cleaved SP-BΔC whereas napsinD283N had lost the catalytic activity. (b) Deglycosylation of wild-type and mutated napsin A proteins. Napsin A and NapsinD283N migrated as 41 kDa proteins and both these proteins migrated as 38 kDa proteins after deglycosylation by endoglycosidase F. Stable HEK293 clones expressing napsin A were analyzed; napsin-1 (expressing wild-type napsin) and clones napsinD283N 21 and napsinD283N 22 (expressing catalytically inactive napsin A). (c) Colocalization of wild-type and mutant napsin A proteins. A vector for wild-type napsin A fused with GFP was transfected into cells expressing HA-tagged napsin A or the napsinD283N mutant. HA-tagged napsin A and the D283N mutant were visualized with Cy3-labeled anti-HA antibody (red). Wild-type napsin A fused with GFP (green) was colocalized with napsinD283N with HA tag (red) (lower panel).
