Las 1000 plus instrument

After the enzyme (0.5 μM) was incubated with the quinones, 10 mM DTT and 4 × Laemmli sample loading buffer (Bio-Rad, CA, USA) were added to the reaction mixture. The resulting sample, along with Precision Plus Protein Dual Color Standard (Bio-Rad), was applied to a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) using 10% TGX™ FastCast™ acrylamide (Bio-Rad) and then migrated by electrophoresis. The proteins in the gels were transferred onto a PVDF membrane (Immobilon-P, Merck) via blotting. The membrane was blocked with EzBlock Chemi (ATTO Co., Tokyo, Japan) for 30 min at room temperature. Subsequently, it was incubated with an anti-3C-like protease (main protease) rabbit polyclonal antibody (1/4000), anti-TD-modified protein monoclonal antibody (1/2500), or an anti-Q5HIAA-modified protein monoclonal antibody (1/2500) in 0.05% Tween® 20 containing Tris-buffered saline, followed by the treatment of the corresponding secondary antibody-HRP conjugate (DAKO). The binding was visualized using Chemi-Lumi One Super (Nacalai Tesque, Inc.). The detection was carried out using a LAS1000 Plus instrument (Fujifilm, Tokyo, Japan). The experiment was repeated twice.