The protein content was determined according to Bradford’s method (Bradford 1976), with bovine serum albumin used as a standard. Protein samples (30 μg) were boiled with 2 Gamma-secretase inhibitor × sample buffer containing 5% β-mercaptoethanol for 5 min, separated by size on 15% polyacrylamide gel under SDS denaturing conditions, and transferred to a nitrocellucose membrane at 90 V for 2 h. The nitrocellulose membranes were stained with ponceau S to assess the efficiency of transfer. Non-specifi c binding was blocked by incubation in block buffer (5% non-fat dry milk, 0.05% Tween-20, 1 × tris-Cl-buffered saline) overnight at 4°C, The membranes were hybridized

with mouse monoclonal antibody recognizing SMAD4 (sc-7966, Santa Cruz Biotechnology, Inc., Santa Cruz, CA),

then incubated with a horseradish peroxidase-labeled goat anti-mouse IgG (1: 500). The bound secondary antibody was detected by enhanced chemiluminescence (Amersham Life Science, Little Chalfont, UK). Housekeeping protein β-actin was used as a loading control. Positive immunoreactive bands were quantified densitometrically (Leica Q500IW image analysis system) and expressed as ratio of SMAD4 to β-actin in optical density units. 2.5 Statistical analysis Caspase inhibition All computations were carried out using the software of SPSS version13.0 for Windows (SPSS Inc, IL, USA). The rank sum test was used to analyze the ranked data. The measurement data were analyzed by one-way ANOVA. Randomized block design ANOVA was used to analyze the statistical difference among different tissue types. In the analysis of glioma morbidity for all patients, we used the Kaplan-Meier estimator and univariate Cox regression analysis to assess the marginal effect of each factor. The differences between Phospholipase D1 groups were tested by log-rank analyses. The joint effect of different factors was assessed using multivariate Cox regression. A Spearman’s analysis was carried out to analyze the correlation between SMAD4

mRNA and protein expression levels. Differences were considered statistically significant when p was less than 0.05. 3. Results 3.1 SMAD4 protein levels in glioma tissues by immunohistochemistry assay and survival analysis SMAD4 expression was studied in a total of 252 glioma specimens of which 113 were low grade glioma (grade I and II) and 139 were high grade (grade III and IV). About 42 specimens taken from normal brain tissue served as control group. Based on immunohistochemistry analysis, positive staining for SMAD4 was mainly observed in the cytoplasm and to a lesser degree in the nuclei of cancer cells. The representative photographs were shown in Figure 1. Among the glioma specimens, 138 (54.8%) glioma specimens were positively stained, and 114 (45.2%) glioma specimens were negatively stained.