Figure 1
From: Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in Arabidopsis
Identification of AtCathB3 in purified fraction containing caspase-3-like activity at pH 5. (a) ECL detection of proteins that interact with biotinylated caspase-3 inhibitor: biotin-DEVD–FMK in total A. th. leaf extract (extract); active fraction purified using bacitracin-sepharose affinity purification (bacitracin). kDa, molecular weight marker. (b) Bacitracin fraction labelled using increasing concentration of biotin-DEVD–FMK, from 0.02 to 10 μM, and after incubation for 1 h at 37 °C the biotinylated proteins were detected by ECL. (c) Capture of the protease of interest. The purified DEVDase fractions were labelled with biotin-DEVD–FMK 1 μM for 1 h at 37 °C and precipitated. After solubilisation, the biotinylated caspase-like protease was captured using streptavidin-agarose magnetic beads. After separation by electrophoresis, the proteins were visualised using silver staining and the major band was cut out and analysed. (d) AA sequence of AtCathB3; the two peptides identified by mass spec in the selected gel slice are underlined, and the catalytic cysteine C131 is in bold and underlined. (e) Phylogenetic tree of the CathB gene family in A. th. AtcathB1: At1g02300; AtcathB2: At1g02305; and AtcathB3: At4g01610 with human cathepsin B (P07858)
