Suc lyamc

Cells (7 × 10⁶/ml) were rinsed, scraped, and homogenized in
extraction buffer (10 mM Tris-HCl, pH 7.5). The homogenate was then centrifuged at
14,000 rpm for 10 min, and 50 to 100 μl of the supernatant, or purified
calpain was assayed for activity. The reaction mixture included (final
concentrations) 200 mM Tris-HCl, pH 8.0, 20 mM CaCl₂, 10 mM cysteine and
50 μM Suc-LYAMC (Sigma) as substrate. Activity was monitored as the change in
fluorescence as a function of time measured at excitation/emission wavelengths of 345
nm and 445, respectively, in a 96-well plate using a Synergy microplate reader.
Purified calpain and its activity was measured using a calpain activity assay kit
(ab65308) as described in the manufacturer's instructions.

The screening of ClpP inhibitors was performed as previously described [21 (link)]. Suc-LY-AMC (Cat# S1153, Sigma–Aldrich) was the specific fluorescence substrate of ClpP, which was used to verify the hydrolytic activity of ClpP and screen the ClpP inhibitor [8 (link),22 (link)]. Briefly, Chinese medicine monomer compounds and ClpP (final concentration 10 µM) were added to ClpP buffer and incubated for 1 h in a black 96-well plate. Then, 100 µM fluorescent substrate was added, and the fluorescence intensity (excitation: 360 nm, emission: 465 nm) was measured using an Infinite M200 instrument (TECAN) after a 1 h incubation at room temperature.