AGS cells (2 × 106 cells/well) were seeded in 6-well plates, pretreated with HECb (12.5, 25, and 50 μg/mL) and a highly selective MEK1 inhibitor (PD98059) at 10 mM for 24 h. Cultures were subsequently infected with H. pylori at a MOI of 300:1 for 1 h. After incubation, cells were lyzed in ice-cold RIPA buffer supplemented with protease cocktail and phosphatase inhibitors (Sigma Fast, 10 mM sodium orthovanadate, 10 mM sodium pyrophosphate, and sodium fluoride 100 mM).
Protein lysates were subjected to SDS-PAGE before being immobilized onto nitrocellulose membranes (Biorad, USA). After transfer, membranes were blocked (20 mM Tris-HCl, pH 7.4, 125 mM NaCl, 0.2% Tween 20, 1% bovine serum albumin, 3% non-fat milk) for 1 h at room temperature and incubated for 4 h at 4°C with specific primary antibodies: p-ERK1/2 and β-actin (as above, Santa Cruz Biotechnology, Dallas, TX, USA). Blots were incubated with secondary antibody rabbit anti-mouse IGG-HRP (sc-358914, Santa Cruz Biotechnology, Dallas, TX, USA) and immunoreactive bands were visualized by chemiluminescence (ECL Amersham, USA) and detected with ChemiDoc XRS system™ software and subsequently analyzed with Image Lab™ (Biorad, Irvine, CA, USA).
Protein lysates were subjected to SDS-PAGE before being immobilized onto nitrocellulose membranes (Biorad, USA). After transfer, membranes were blocked (20 mM Tris-HCl, pH 7.4, 125 mM NaCl, 0.2% Tween 20, 1% bovine serum albumin, 3% non-fat milk) for 1 h at room temperature and incubated for 4 h at 4°C with specific primary antibodies: p-ERK1/2 and β-actin (as above, Santa Cruz Biotechnology, Dallas, TX, USA). Blots were incubated with secondary antibody rabbit anti-mouse IGG-HRP (sc-358914, Santa Cruz Biotechnology, Dallas, TX, USA) and immunoreactive bands were visualized by chemiluminescence (ECL Amersham, USA) and detected with ChemiDoc XRS system™ software and subsequently analyzed with Image Lab™ (Biorad, Irvine, CA, USA).