Sb203508

Activated phospho-ERK, phospho-p38 and phospho-JNK were determined by instant one ELISA kit (eBiosciences) as per the manufacturer's instructions. Briefly, after 1 h post-infection, Ham's-F12 medium was removed and cells were washed with 1× PBS. After washing, cells were lysed with 300 μl of freshly prepared lysis buffer (provided in kit) with shaking (∼300 rpm) at room temperature for 10 min. 50 μl of the prepared sample lysate was incubated with 50 μl of antibody cocktail (capture and detection antibody) for 1 h at room temperature. After incubation, cells were washed three times with 1× wash buffer and 100 μl of detection reagent was added into the wells for 20 min at room temperature in dark. 100 μl of stop solution was added into each well and absorbance of the samples was measured using Tecan ELISA plate reader at 450 nm. For p38 and JNK inhibition experiments; AGS cells were pretreated with SB203508 (p38 inhibitor) and SP600125 (JNK inhibitor) at the indicated final concentration (both from Sigma-Aldrich) for 1 h prior to TieA treatment. As a negative control, AGS cells were treated with dimethyl sulfoxide alone. For cytokine analyses (IL-8, IL-6, IL-1β and TNF-α), ready set Go ELISA kit (eBiosciences) was used as per the manufacturer's protocol.