Human 26s proteasome protein

UPS activity was measured as previously described (Brehm et al., 2015 (link)). Briefly, erythroblasts were gently lysed in TSDG buffer (10 mM Tris-HCl, 1.1 mM MgCl2, 10 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 2 mM ATP, 10% (v/v) glycerol, pH 7.0) with 3 freeze-thaw cycles to prevent UPS denaturation. 10 μg cell of lysates were assayed for protease activity in assay buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 2 mM ATP) with either 100 μM Suc-Leu-Leu-Val-Tyr-aminomethyl-coumarin (Suc-Leu-Leu-Val-Tyr-AMC, Enzo Life Sciences) for chymotryptic-like activity, BZ-Val-Gly-Arg-AMC (Enzo Life Sciences) for tryptic-like activity, or Z-Leu-Leu-Glu-AMC (Enzo Life Sciences) for caspase-like activity. UPS activity was determined by measuring, over time, the fluorescence of AMC liberated from peptides substrate (Ex/Em: 380nm/460nm). For purified UPS activity, 2 μg of Human 26S Proteasome Protein (R&D Systems) were incubated, in TSDG buffer, with the indicated concentrations of sodium acetate, sodium succinate, sodium malonate, sodium L-lactate, L-lactic acid or with epoxomicin (10 μM; Sigma–Aldrich) for 30 min at room temperature. The chymotryptic-like activity was then measured as described above.

Recombinant human HtrA2 (aa 134–458) and human 26S Proteasome Protein were purchased from R&D Systems (1458-HT-100 and E-365). Recombinant Tau protein (2N4R) was obtained from rPeptide (T-1001-2).