Pepstatin

Synthetic peptides were produced by solid phase synthesis, purified by liquid chromatography, and confirmed by mass spectrometry (Tufts University Core Facility). Peptides were dissolved in phosphate buffered saline (PBS) at a concentration of 1 mM, as measured using optical absorbance measurements at 280 nm. Cell culture media was obtained from Corning. All cell lysis buffers were supplemented with protease inhibitors comprised of AEBSF (0.5 mM, Santa Cruz, SC-202041B), bestatin (0.01 mM, Fisher/Alfa Aesar, J61106-MD), leupeptin (0.1 mM, Santa Cruz, SC-295358B), and pepstatin (0.001 mM, Santa Cruz, SC-45036A), and cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche) as required. MG132 was obtained from Cell Signaling Technologies.

CD38 cyclase activity was measured according to [75 (link)] with minor changes. MOLM-13 cells were sonicated with 40W 20 kHz 3 times for 5 s on ice in a lysis buffer containing 10 mM Tris/HCl (pH 7.4), 0.25 M sucrose, 20 mM NaF, 1 mM DTT, 5 mM EDTA, 1 mM PMSF (all chemicals from Sigma–Aldrich), and a protease inhibitor cocktail (chymostatin, leupeptin, antipain, and pepstatin; Santa Cruz Biotechnology, sc-24948A). Fluorescence (ex = 300 nm, em = 410 nm) of reaction mix containing 1 × 10⁶ lysed MOLM-13, 200 μm nicotinamide guanine dinucleotide sodium salt (NGD) and 0 to 1.28 μm 78c was recorded for 1 h. To generate dose-response curve, normalized relative fluorescence intensity at 1 h was plotted against log 78c concentration.