Genegnome hr system

Exponentially growing yeast cells were harvested. Cells were washed and resuspended in ice-cold Buffer 1 supplemented with protease inhibitors as described above. Cells were broken in a bead-beater by glass beads. Extract was centrifuged at 4 °C. Supernatant was boiled with SDS-gel loading buffer and subjected to Western blotting by using anti-FLAG and anti-Tubulin (T6074, Sigma) antibodies. The blots were scanned and quantified by GeneGnome HR system (Syngene, Cambridge, UK).

MNK28 cells, SGC7901 cells, MNK28 cells transfected with GV268-miR-588, and SGC7901 cells transfected with GV268-miR-588 were harvested. For immunoblotting, cells were lysed by radioimmunoprecipitation assay (RIPA) buffer (P0013C, Beyotime) and centrifugated at 10,000 × g for 10 minutes. Extracted proteins were then resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Protein blots were incubated with appropriate antibodies, and protein bands were visualized by enhanced chemiluminescence (ECL) or ECLPLUS (Amersham, Piscataway, NJ). The antibodies used were CXCL5 (D263012-0025, BBI), CXCL9 (ab202961, Abcam), CXCL10 (D220389-0025, BBI), and antitubulin (#T8203, Sigma-Aldrich, St. Louis, MO). The GeneGnome HR system (Syngene, Cambridge, UK) was used to scan blots.

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).