Expression of plasminogen activator inhibitor type 1 (PAI-1) mRNA and protein in mixed glial cultures. (A) Cell lysates or conditioned medium were prepared from either untreated or lipopolysaccharide (LPS)/interferon (IFN)-γ-treated mixed glial cultures (24 hours treatment). Western blotting analysis was conducted to evaluate the expression level of the 47 kDa PAI-1 protein. Ponceau S staining images are shown for comparison. (B) The mixed glial cells, microglia, and astrocytes were stimulated with a combination of LPS (100 ng/ml) and IFN-γ (50 U/ml) for 24 hours, and conditioned medium were collected. PA1-1 secretion was measured by ELISA. Results are given as mean ± SD (n = 3). *P < 0.05, ** P < 0.01. (C, D) Real-time PCR was performed to detect PAI-1 mRNA expression in glial cells treated for 6 hours with LPS (100 ng/ml) and IFN-γ (50 U/ml) either alone or in combination as indicated. GAPDH was used as an internal control. Results are given as mean ± SD (n = 3). *P < 0.05, ** P < 0.01, NS = not significant; compared with the untreated control cells.