Expression of GR and Brca1 is decreased in cells stably expressing an shRNA vector against endogenous GR. EPH-4 cells were stably transfected with a puromycin selectable marker and either an empty vector (H1-2; EV) or an shRNA vector directed against the endogenous glucocorticoid receptor (shGR). Cells were puromycin-selected and expanded. A. EV-50, shGR-19, and shGR-73 stable clone lines were lysed and subjected to Western blotting to determine GR expression (shown in left panel). Densitometric analysis was performed to quantify the level of GR protein knockdown in shGR-73 and shGR-19 relative to EV-50 (shown in right panel; numbers indicate protein levels relative to EV-50). B-C. RNA was prepared from EPH-4 stable cell lines EV-50, shGR-73, and shGR-19, and qRT-PCR analysis of mouse B. Nr3c1 (GR) and C. Brca1 expression was conducted using TaqMan gene expression assays for each gene. Raw C
t values for each gene were normalized to raw C
t values for mouse Tbp internal control for triplicate samples, and are presented as the level of expression relative to the EV-50 sample. Bars represent the mean of technical replicates, and error bars represent standard deviation (N = 3). Statistically significant changes in gene expression relative to EV-50 are indicated for each gene: one asterisk, p < 0.05 (significant); two asterisks, p < 0.005 (very significant).