Adenoviral siRNA expression leads to reduction of the target protein. (A) Protein expression levels for CHUK (IKKα), IKBKB (IKKβ), and α-tubulin were detected by Western analysis of samples derived from U2OS cells 6 d after infection with Ad-siRNA-CHUK, Ad-siRNA-IKBKB-1, control virus Ad-siRNA-GL2, or from uninfected cells (no virus). The protein levels of CHUK and IKBKB were specifically reduced by the corresponding adenoviral knock-down construct. (B) The effect of adenoviral knock-down was determined at the level of protein activity. Gelatin-degrading activity of MMP2 was measured using 10% gelatin-zymogram gels. Supernatants of primary synoviocytes were analyzed 6 d after infection at m.o.i. 7500 with Ad-siRNA-MMP2-1 and Ad-siRNA-MMP2-3, and compared with Ad-siRNA-control, a virus containing a target sequence against M6PR. For reference, an adenoviral cDNA expression construct encoding the MMP2 protein (Ad-MMP2) was used and demonstrated the identity of the MMP2 band. The inverted image of the zymogram is presented. (C) Knock-down of GαS (encoded by GNAS) by Ad-siRNAGNAS results in reduction of β2-adrenergic receptor signaling. HEK293 cells were infected (m.o.i. 150) with Ad-siRNA-GNAS or control virus Ad-siRNA-empty for 3 d. Subsequently, the medium was refreshed and the cells were reinfected (m.o.i. 300) with a reporter adenovirus encoding a luciferase gene under control of the cAMP-responsive element (CRE)-dependent promoter and incubated for 2 extra days. The endogenously expressed β2-adrenergic receptor is stimulated with increasing concentrations of the agonist isoproteranol. Signaling via the active GαS subunit results in increased cAMP levels and subsequently activates the CRE-dependent promoter. The luciferase activity (indicated on the Y-axis as relative light units; RLU) was measured 6 h after stimulation with the agonist. The isoproteranol concentration (in moles per liter) is plotted as logarithmic values on the X-axis. pEC50 values remain largely unaffected (values between 8.2 and 8.5). Error bars indicate the standard deviation values based on 3 data points.
