Figure 2.

BAG6 Is Cleaved by Human Caspase-1 in Vitro and by Pant Caspase-1-Like Protease Activity in Vivo.

(A) Structure of BAG6, showing the conserved BAG domain, nuclear localization signal (NLS) and calmodulin binding motif (CaM). The arrow indicates the amino acid position (amino acid 760) cleaved by the caspase-1 protease. aa, amino acid.

(B) Arabidopsis BAG6 is cleaved by human caspase-1 in vitro. HA-tagged full-length BAG6 and BAG6^D760A under 35S promoter were transiently expressed in N. benthamiana for 3 d. Equal amounts of purified HA-BAG6 (10 μg) were treated with recombinant human caspase-1 (2 units) at 37°C for 2 h and analyzed by immunoblotting with anti-HA antibody. CBS, Coomassie blue staining

(C) BAG6 is cleaved by plant caspase-1-like protease activity in vivo. Proteins were extracted from Arabidopsis plants expressing HA-BAG6 and HA-BAG6^D760A under the 35S promoter or native promoter in response to B. cinerea 24 h after inoculation and subjected to immunoblotting using anti-HA antibody (left panel). Proteins from leaves with or without lesion mimics in Arabidopsis plants expressing HA-BAG6 under 35S promoter were monitored for BAG6 cleavage using anti-HA antibody (right panel). Equal loading was confirmed by SDS-PAGE and Coomassie blue staining.