Inhibition of cathepsin S-mediated proteolytic cleavage of MHC2 associated invariant chain in mouse splenocytes assessed as accumulation of lip10 by Western blot analysis
Inhibition of cathepsin S-mediated proteolytic cleavage of MHC2 associated invariant chain in mouse splenocytes assessed as induction of accumulation of lip10
Inhibition of cathepsin S-mediated proteolytic cleavage of MHC2 associated invariant chain in mouse splenocytes assessed as induction of accumulation of lip10 at 10 nM
Inhibition Assay: First, spleen cells collected from C57BL/6 mice were seeded in a 96-well plate so as to be a 1× 105 cells/well, then, 5-fold dilution of 10 mM DMSO solution of the test compound was serially performed with RPMI 1640 medium (including 10% fetal bovine serum (FCS), 5×10^−5 M 2-mercaptoethanol, 50 IU/mL penicillin and 50 μg/mL streptomycin) nine times such that the final concentration becomes 0.026 nM to 10 μM, and the resultant products were added thereto (the final DMSO concentration was 0.1%). At the same time, LPS (Sigma L4005) was added to the wells such that the final concentration becomes 2 μg/mL, and culture was performed at 37° C. for 48 hours in 5% CO2. After culturing, the cells were stained at 4° C. for 20 minutes with PE-labeled streptavidin (BD BIOSCIENCE 554061) and biotin-labeled YAe antibody (EBIOSCIENCE 13-5741-85) together with FITC-labeled anti-mouse B220 antibody (BD BIOSCIENCE 553088), and an expression level (fluorescence intensity of YAe-bioti
Inhibition of cathepsin S in C57/Bl6 mouse spleen lysate using Z-Val-Leu-Arg-AMC as substrate at 100 uM preincubated for 30 mins followed by substrate addition
Inhibition of cathepsin S in C57/Bl6 mouse spleen lysate using Z-Val-Leu-Arg-AMC as substrate at 10 uM preincubated for 30 mins followed by substrate addition