This suggested that ERK activation is causally involved in driving prolif

All PCR reactions were performed in triplicate, with the mean being used to determine mRNA levels. Relative mRNA expression levels for MMP 26 were determined using the 2 CT method and normalized to the endogenous refer ence gene, GAPDH. Western Blotting analysis Total protein was isolated from B6Tert 1, JEG 3 or CTB cells using lysis buffer. The protein extracted from aT3 Janus キナーゼ 阻害剤 1 cells was kindly provided by Dr. Peter C. K. Leung in University of British Columbia, Canada. Total protein was run on 10% SDS polyacrylamide gels. After electrotransferring the protein to the nitrocellulose membrane, the membrane was immuno blotted using specific primary antibodies for MMP 26, GnRHR, phospho ERK12 or phospho JNK at 4 C over night.<br><br> The signals were detected with horseradish perox idase conjugated secondary antibody for 1 hour and visualized using the enhanced chemiluminescence sys tem. After stripping, 価格 LDE225 the membranes were reprobed with b actin, total ERK12 and total JNK antibodies, respectively. The relative den sity of MMP 26 was determined by normalization to the density value of b actin. The relative densities of phos phorylated forms of ERK12 and JNK were normalized to total values of ERK12 and JNK. All densities were analyzed using the Gel Pro Analyzer. Transwell insert invasion assay In vitro cell invasion was assayed by determining the ability of cells to invade a synthetic basement mem brane. Briefly, 24 well fitted transwell inserts with mem branes were coated with growth factor reduced Matrigel at a concentration of 200 ug mL and placed in a 24 well plate.<br><br> B6Tert 1 cells at a concentration of 2 104 were seeded in each insert containing defined medium supplemented with 1 ngml EGF and 1 mgml insulin and lower chambers were loaded with defined medium containing 10 ngml EGF and 10 mgml insulin. After incubating with or without GnRH I or LY2157299 700874-72-2 GnRH II for 24 h, the cells were then fixed and stained with crystal violet. Non invaded cells on the upper surface of the membrane were removed using a cotton swab. The membranes were cut from inserts and mounted onto glass slides. The number of stained cells was counted, in at least 15 randomly selected non overlapping fields of the membranes, using a light microscope. Statistical analysis Data were shown as the mean SEM of three individual experiments performed in duplicate or triplicate.<br><br> Statis tical analysis was carried out using one way ANOVA followed by Dunnetts test, and differences were consid ered significant for P 0. 05. Representative images of Western blot are shown. Results The characteristics of B6Tert 1 cells The expression of GnRHR mRNA has been shown in JEG 3 cells and first trimester CTB cells in primary cul ture. To determine whether the GnRHR gene is expressed in B6Tert 1 cells, levels of GnRHR protein were assessed by Western blotting. As shown in Figure 1A, the expression of GnRHR protein was present in both B6Tert 1 and JEG 3 cells. Mouse pituitary gonado trope aT3 1 cells, previously shown to express GnRHR, were used as the positive control. To characterize the invasiveness of B6Tert 1 cells in response to GnRH I and II, cells treated with the native peptides of these two subtypes of hormone were sub jected to invasion assay.