Western blotting for p38, p p38, JNK and p JNK Western blotting for the e pression of p38, p p38, JNK and p JNK in AGS or MKN 45 cells was conducted using previously described methods. The dilution of pri mary antibodies used was as followings rabbit anti human p38, p p38, JNK or p JNK. Anti B actin was used as a control for the Western blots. Cell migration and invasion assay For the invasion assay of AGS or MKN 45 cells, we used Sumida Ts and our previous methods. Millicell Hanging Cell Invasion Chambers with 8 um pore filter were coated with 12 uL of ice cold Matrigel. AGS or MKN 45 cells were added to the upper chamber of these matrigel chambers in 200 ul serum free F12 or DMEM medium with or without 20 ng ml human IL 1B. Cells were then placed into 24 well plates in F12 or DMEM medium containing 10% FBS.

To evaluate the role of the SB202190 or SP600125 or BiPS inhibitor, cells were pre treated with the reagent for 3 h, and the stimulations were then performed. To evaluate the role of p38 siRNA or JNK siRNA or MMP2 siRNA or MMP9 siRNA or MMP2 siRNA plus MMP9 siRNA in cell migration and invasion, AGS or MKN 45 cells were transfected with scrambled siRNA or p38 siRNA or JNK siRNA or MMP2 siRNA or MMP9 siRNA or MMP2 siRNA plus MMP9 siRNA for 36 h. Following this, the transfected cells were seeded at a density of 5 104 per well and then in 200 ul of serum free medium for the stimulation. When the 20 h incubation was completed, cells were fi ed with methanol and stained with Giemsa or crystal violet. Cotton tips were used to remove the cells that remained in the matrigel or attached to the upper side of the filter.

Light microscopy was used to count the Carfilzomib cells on the lower side of the filter. The assays were performed in duplicate, and the results were then averaged. The methods used for the migration assay were almost the same as for the invasion assay described above, e cept no matrigel was used to coat the well and the incubation time was 15 h. RT PCR assay RT PCR for amplification of human MMP2, MMP9, c fos, p38 used the methods described by us previously. Total RNA was e tracted from AGS or MKN 45 cells or mouse lung metastatic human gastric cancer cell MKN 45 with the Trizol reagent. The e pression levels of human MMP2, MMP9, c fos, p38 and GAPDH mRNA were detected by first reverse transcribing the total RNA, followed by PCR with the following primers MMP 2 and 9 zymography assay MMP 2 and 9 zymography assay��MMP 2 and 9 protease activities in the concentrated supernatant medium of AGS or MKN 45 cells were detected by zymography. Briefly, 8% SDS PAGE containing gelatin zymogram gels were used to separate the proteins with electrophoresis.