GSK3α inhibition up-regulates IL-12p40 expression in BEC stimulated with PGN.

<p>A) BEC were left untransfected (-), transfected with control siRNA (siRNA control), transfected with siRNA targeting GSK3α (siRNA GSK3α1) or transfected with siRNA targeting GSK3β (siRNA GSK3β1). B) BEC were left untransfected and unstimulated (-), stimulated with 10 μg/mL of PGN for 4 h, transfected with control siRNA (siRNA control), transfected with control siRNA and then stimulated with 10 μg/mL PGN for 4 h, transfected with siRNA targeting GSK3α (siRNA GSK3α1) or transfected with siRNA targeting GSK3α (siRNA GSK3α1) and then stimulated with 10 μg/mL of PGN for 4 h. C) BEC were left untransfected and unstimulated (-), stimulated with 10 μg/mL of PGN for 9 h, transfected with control siRNA (siRNA control), transfected with control siRNA and then stimulated with 10 μg/mL of PGN for 9 h, transfected with siRNA targeting GSK3α (siRNA GSK3α1) or transfected with siRNA targeting GSK3α1 and then stimulated with 10 μg/mL of PGN for 9 h. Protein extracts were analyzed by western blot and probed with a monoclonal antibody against the phosphorylated forms of GSK3α and GSK3β (A). To verify that equal amount of proteins was loaded in each lane, blots were stripped and reprobed with an antibody that recognizes the nonphosphorylated form of β-actin (A). Blots are representative of three independent experiments. Total RNA was extracted and relative transcript level of IL-12p40 was quantitated by qRT-PCR, using the delta-delta Ct method and amplification of β-actin as a reference gene (B). Cell-free supernatants were analyzed by ELISA for production of IL-12p40 (C). Results are expressed as the mean ± S.E.M. (<i>n</i> = 3). *<i>p</i> <0.05; **<i>p</i> <0.01. All data were compared with the untreated and untransfected controls.