Cathepsin b activity assay kit

The C-terminal modified RR peptide (Arg-Arg ethylamide, RR-NHEt; 357.4 Da) was synthesized by ANYGEN (Gwangju, Republic of Korea). Acetonitrile (ACN), antibiotic antimycotic solution, a cathepsin B activity assay kit, N,N-diisopropylethylamine (DIPEA), anhydrous N,N-dimethylformamide (DMF), Dulbecco’s Modified Eagle’s Medium (DMEM), diethyl ether, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), methanol, phosphate-buffered saline (PBS), trifluoroacetic acid (TFA), and ursodeoxycholic acid (UDCA) were purchased from Sigma-Aldrich (Milwaukee, WI, USA). Fetal bovine serum (FBS) was obtained from Gibco (Waltham, MA, USA). An Ez Cytox kit was obtained from DoGenBio (Seoul, Republic of Korea). Neutral buffered formalin (10%) was purchased from HuBenTech (Damyang, Republic of Korea). A Pierce BCA protein assay kit was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Chloroform was purchased from DAEJUNG (Siheung, Republic of Korea).

Mouse colorectal cancer (CT26) cells were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea). They were cultured in 10% FBS containing high-glucose DMEM medium and then collected by scraping, with treatment of cell lysis buffer, using the cathepsin B activity assay kit (Sigma-Aldrich, Milwaukee, WI, USA). The cell solution was centrifuged at 13,000 rpm for 15 min at 4 °C, followed by 5 min incubation on ice for the collected supernatants. After determining the protein concentration for the collected supernatant using a Pierce BCA protein assay kit (Thermo Fisher Scientific), 50 µg of cell lysate was combined with cathepsin B reaction buffer (cathepsin B activity assay kit) in a 96-well black plate. Cathepsin B inhibitor (cathepsin B activity assay kit, 1 mM) was used as a control to inhibit cathepsin B activity in CT26 cells and for comparison with different treatments of RR–BA (1, 5, or 10 mM). In addition, RR peptide (1, 5 or 10 mM) was also used to compare the inhibition effect of cathepsin B activity with RR–BA. After the addition of a fluorogenic cathepsin B substrate, Ac-RR-AFC (cathepsin-B-specific substrate in the cathepsin B activity assay kit, 1000 µM), the plate was incubated for 2 h at 37 °C. Then, the fluorescence of the wells was measured using a microplate reader (SpectraMax M2) at wavelengths of λ_Ex = 400 nm/λ_Em = 505 nm.