Assays of caspase-9 and caspase-3 activity were carried out by using caspase-9 assay kit (abcam, ab65607) and caspase-3 assay kit (abcam, ab39383) according to the manufacturer's protocol as described previously [40 (link)].
Assays of cathepsin B (Cat-B) and cathepsin D (Cat-D) activity were carried out by using cathepsin B assay kit (abcam, ab65300) and cathepsin D assay kit (abcam, ab65302) according to the manufacturer's protocol. Briefly, 50 μg of cytosolic fraction protein prepared from control or stimulated cells was added up to the final reactive to 200 μL per well in a 96-well plate. Aliquots of assay volume were treated with 140 mM site-specific substrates in assay buffer at 37°C with 10 mM DTT for 0.5 h. Cleavage of the preferred Cat-B substrate [sequence RR labeled with AFC (amino-4-trifluoromethyl coumarin)] and Cat-D substrate [sequence GKPILFFRLK(Dnp)-D-R-NH2, labeled with MCA] by Cat-B and Cat-D release AFC and MCA respectively. The AFC fluorescence is measured at 400 nm excitation and 505 nm emission, and the MCA fluorescence is measured at 328 nm excitation 460 nm emission with a VersaFluor Fluorometer (Bio-Rad, Hercules, CA) respectively. The relative fluorescent units (RFU) were normalized with protein concentrations.
Assays of cathepsin B (Cat-B) and cathepsin D (Cat-D) activity were carried out by using cathepsin B assay kit (abcam, ab65300) and cathepsin D assay kit (abcam, ab65302) according to the manufacturer's protocol. Briefly, 50 μg of cytosolic fraction protein prepared from control or stimulated cells was added up to the final reactive to 200 μL per well in a 96-well plate. Aliquots of assay volume were treated with 140 mM site-specific substrates in assay buffer at 37°C with 10 mM DTT for 0.5 h. Cleavage of the preferred Cat-B substrate [sequence RR labeled with AFC (amino-4-trifluoromethyl coumarin)] and Cat-D substrate [sequence GKPILFFRLK(Dnp)-D-R-NH2, labeled with MCA] by Cat-B and Cat-D release AFC and MCA respectively. The AFC fluorescence is measured at 400 nm excitation and 505 nm emission, and the MCA fluorescence is measured at 328 nm excitation 460 nm emission with a VersaFluor Fluorometer (Bio-Rad, Hercules, CA) respectively. The relative fluorescent units (RFU) were normalized with protein concentrations.
Free full text: Click here
