Anti flag monoclonal

We prepared sonicated DNA samples from AGS and MKN7 cells using a ChIP-IT Express (No. 39163, Active Motif), and then incubated with 1-3 μg of three Histone H3 methylation-related (H3K4me3, No. 39915; H3K9me3, No. 29766; and H3K27me3, No. 39155; Active Motif) and two histone acetylation-related antibodies (acetyl-Histone H3 (K9 and K14), #06-599, acetyl-histone H4 (K5, K8, K12, K16), #06-866; Upstate). Moreover, after transfection of the SET7/9 expression vector or its siRNA into GC cells, ChIP assays were conducted using anti-H3K4me1 polyclonal (ab8895, Abcam, Cambridge, UK) and anti-FLAG monoclonal (Sigma-Aldrich Japan) antibodies. Histone H3 (No. 39163, Active Motif) and Normal Rabbit IgG (No. 2729, Cell Signaling Technology) were used as positive and negative controls, respectively. Input DNA samples were used as internal controls. The primer sequences and their conditions are shown in Supplementary Table S2.

V. parahaemolyticus harboring pBAD-vtrA, pBAD-vtrA-FLAG or their derivatives were grown in Luria-Bertani (LB) media containing 0.5% NaCl in the presence of 0.01% (w/v) L-arabinose to an OD600 of 2.0. Then, the bacterial cells were harvested by centrifugation at 3,000 × g for 15 min. The pellets were re-suspended with 200 μl of the periplasting buffer (200 mM Tris-HCl at pH 7.5, 20% sucrose, 1 mM EDTA, and 20 mg/ml lysozyme) and incubated for 5 min at room temperature. The cells were osmotically shocked by the addition of 200 μl of ice-cold distilled water and incubated for 5 min on ice. After centrifugation at 12,000 × g for 2 min, the spheroplasts were treated with 500 U/ml benzonase for 5 min at room temperature, and sonicated to induce lysis. The sample was centrifuged twice at 12,000 × g for 2 min to remove unlysed cells. The supernatants were centrifuged at 100,000 × g for 1 h, and the resulting supernatants were collected as the cytoplasmic fraction. The membrane fractions were dissolved in SDS loading buffer. Each fraction was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis using anti-HA monoclonal (Medical & Biological Laboratories, Nagoya, Japan), anti-FLAG monoclonal (Sigma-Aldrich, St. Louis, MO, USA), anti-DnaK and anti-OmpA antibodies.