Western Blot Analysis of Protein Samples

Western blotting was performed as described previously [23 (link),26 (link),27 (link)]. Briefly, a cell pellet was resuspended in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, and cOmplete™ Protease Inhibitor Cocktail tablet (Roche, Basel, Switzerland) per 25 mL buffer). Samples were incubated for 30 min on ice followed by 30 s sonication (Bandelin Sonopuls mini20 homogenizer). The samples were then centrifuged for 15 min (12,000× g at 4 °C). The cleared lysates were separated and analyzed by SDS-PAGE (NuPage^® 10% bis-tris gel) with subsequent blotting onto a PVDF-membrane. The blotted membrane was blocked using blocking buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 3% bovine serum albumin (BSA), 0.5% Tween 20) overnight at 4 °C. Immunodetection was conducted using mouse anti-His₆ and HRP-conjugated rabbit anti-mouse antibodies (IBA) (1:2500 dilution). Band quantification was performed using ImageJ [28 (link)].