P30266

pZP32 and pZP33 were transformed into FIM-1∆U. Transformants were grown in YG medium and 3 × 10⁸ cells were collected after indicated times. Cells were washed and resuspended in 400 μL lysis buffer (50 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate) supplemented with protease inhibitors cocktail (05892970001, Roche Applied Science). Cells were mixed with 400 μL acid-washed glass beads (G8772, Sigma-Aldrich, Missouri, USA) and processed by a bead-beater (FastPrep-24, MP, California, USA) at 6 m/s for 2 min. Lysate was centrifuged at 13,200 rpm for 20 min at 4°. 100 μL Supernatant was supplemented with 25 μL 5XSDS PAGE loading buffer (150 mM Tris–HCl (pH 7.0), 12% SDS, 6% 2-mercaptoethanol, 30% glycerol (V/V), 0.05% Coomassie Brilliant Blue G-250) and boiled. 10 μL Samples were subjected to Western blot assay [28 (link)]. Anti-His Tag antibody (1:5000 dilution) (M30111, Abmart, Shanghai, China), anti-histone H3.1 antibody (1:3000 dilution) (P30266, Abmart) and horseradish peroxidase conjugated goat-anti-mouse secondary antibody (1:3000 dilution) (074–1806, KPL, USA) were used in the Western blot. The blots were visualized by ECL prime Western blotting detection reagent (RPN2232, GE Healthcare, Illinois, USA) and scanned by GeneGnome HR system (Syngene, Cambridge, UK).