Briefly, 12% SDS PAGE was used to detect selleck chemical Rucaparib the proteins. After the proteins were transferred onto PVDF membranes and incubated with the Inhibitors,Modulators,Libraries rabbit anti human ERK1 2 or p ERK1 2 or mouse anti human MMP 9 anti body. The primary antibody was detected by a horseradish peroxidase conjugated goat anti rabbit or mouse secondary antibody. The immunoreactive protein bands were visualized with enhanced chemiluminescent. Anti B actin was used as a control for the Western blots. Cell migration and invasion assay For the invasion assay of BT474 cells, we used methods described by Sumida et al.Millicell Hanging Cell Invasion Chambers with 8 um pore filter were coated with 12 uL of ice cold Matrigel. BT474 cells were added to the upper chamber of these matrigel chambers in 200 ul serum free RPMI 1640 medium with 20 ng ml human EGF, 20 ng ml IL 1B, and both or nei ther.

Cells were then placed into 24 well plates in RPMI 1640 medium containing 10% FBS. To evaluate the role of the U0126 inhibitor, cells were pre treated with the re agent for 3 h, and the stimulations were then performed. To evaluate the role of ERK1 2 siRNA in cell migration and invasion, BT474 cells were transfected with scrambled siRNA or ERK1 2 siRNA for 36 Inhibitors,Modulators,Libraries h. Following this, the transfected cells were seeded at a density of 50,000 per well and then in 200 ul of serum free medium for the stimulation. When the 22 h incubation was completed, cells were fixed with methanol and stained with Giemsa. 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 cells on the lower Inhibitors,Modulators,Libraries 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, ex cept no matrigel was used to coat the well and the incu bation time was 16 h. RT PCR assay Inhibitors,Modulators,Libraries Total RNA was extracted from BT474 cells with the Trizol reagent. The expression levels of MMP 9 mRNA were detected by first reverse transcribing the total RNA, MMP 9 zymography assay MMP 9 protease activities in the concentrated super natant medium of BT474 cells were detected by zymo graphy. Briefly, 8% SDS PAGE containing gelatin zymogram gels were used to separate the proteins with electro phoresis. Renaturing and developing the gels were per formed according to the manufacturers instructions, and the gels were then stained with Coomassie blue. AP 1 luciferase reporter gene assay BT474 cells were transfected with AP 1 luc vector or AP 1 luc vector plus scrambled siRNA or ERK1 2 siRNA with Lipofectamine 2000. B gal Inhibitors,Modulators,Libraries plasmid was co transfected with AP 1 reporter selleck chemicals llc plasmids to serve as the control for transfection efficiency.