Complete edta free

Cell pellets from 1 L LB culture were suspended with 30 mL of buffer A (20 mM Tris-HCl (pH 7.9), 300 mM NaCl, 20 mM imidazole, 5 mM beta-mercaptoethanol (BME), 1% Tween20) supplemented with a tablet protease inhibitor cocktail (complete, EDTA-Free, Sigma). Cells were lysed by sonication. Soluble fractions were separated by centrifugation (20,000 × g for 40 min at 4 °C). The supernatant was loaded on a pre-equilibrated 1 mL HisTrap column (GE Healthcare, USA) with buffer B (20 mM Tris-HCl (pH 7.9), 300 mM NaCl, 20 mM imidazole) and washed with 10 column volumes (CV) of buffer B and 50 CV of buffer C (20 mM Tris-HCl (pH 7.9), 300 mM NaCl, 50 mM imidazole), and eluted with buffer D (20 mM Tris-HCl (pH 7.9), 300 mM NaCl, 300 mM imidazole). Eluent was injected to HiLoad 26/600 superdex 200 pg (GE Healthcare, USA) column equilibrated with buffer E (20 mM HEPES pH 7.5, 300 mM NaCl, 10% Glycerol, 0.5 mM TCEP). The major fractions were analyzed with 4–20% SDS-PAGE Gel stained with coomassie brilliant blue. Protein concentrations were determined by Bradford protein assay kit II (Bio-Rad, USA) with bovine serum albumin as the standard.

Cells from Escherichia coli strain Rosetta2 (DE3; Merck Millipore, Japan) were transformed with pDEST15-GST-Steroleosin B and pre-cultured overnight in 3-ml of LB medium at 37 °C. Flasks containing 50 ml of LB medium supplemented with 50-µg/ml kanamycin were inoculated with 500 µl aliquots of saturated culture. Cells were cultivated until the optical density at 600 nm (OD₆₀₀) reached 1.0. The cultures were then supplemented with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and cultured for a further 3 h at 23 °C. Next, the cells were harvested by centrifugation at 10,000 × g for 10 min at 4 °C. After removal of supernatant, pellets were stored at −80 °C until purification of GST-fused Steroleosin B, as described elsewhere. Briefly, cell pellets were re-suspended in 15 ml aliquots of ice cold extraction buffer containing 1× PBS, 0.1% β-mercaptoethanol and 1× Complete EDTA-free (Sigma-Aldrich, Japan) and then ultrasonically lysed on ice. Homogenates were finally centrifuged at 10,000 × g for 15 min at 4 °C before being subjected to the pull-down assays using SIB, as described above.

Isolated corpus and antrum gastric glands were lysed using ice-cold lysis buffer (25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% Na-Deoxycholate, 0.1% SDS, 1 mM EDTA) with a protease inhibitor cocktail (Complete EDTA free, Sigma-Aldrich) and rotated at 4 °C for 1 h. Lysate was centrifuged at 4 °C for 20 min, and the supernatant was used for western blotting. The protein concentration of the supernatant was measured by Bradford protein assay. The supernatant was mixed with sample buffer and incubated at 95 °C for 5 min. Samples were separated on SDS-PAGE gels and transferred to Nitrocellulose membranes. The membranes were blocked by Blocking One (Nacalai) at room temperature for 1 h and incubated with primary antibodies overnight at 4 °C. The membranes were washed with TBST and incubated with HRP-conjugated secondary antibody at room temperature for 1 h. After washing with TBST, the membranes were incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and imaged using a gel imager (LAS-4000, Fujifilm, Tokyo, JAPAN).