FIGURE 8.
Reduction of IL-18Rα in human PBMC. PBMC from 10 healthy volunteers were transfected with concentration-matched pairs of 250 nm siRNA to IL-18Rα or scrambled siRNA. A, cells were stimulated with 1 μg/ml LPS or 25 ng/ml IL-12 and 50 ng/ml IL-18. Supernatants were harvested after 20 h, and cytokine levels were determined by multiplex or regular ELISA. Means ± S.E. of absolute cytokine concentrations from 7 of the 10 donors are shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001 for siIL-18Rα versus scrambled. B, cells from four donors were lysed 2 and 20 h after stimulation. RNA was isolated and subjected to reverse transcription and quantitative real time PCR. Changes in mRNA expression were calculated using the ΔΔCT method. Scrambled controls were set at 100% for each condition; percentage changes in IL-18Rα mRNA levels normalized to β-actin and glyceraldehyde-3-phosphate dehydrogenase are shown. C, lysates from the same experiment were subjected to Western blot analysis with staining for SOCS3. One representative of three blots giving similar results is shown. The bands depicted are from one gel; between the pair, one lane containing a condition not pertaining to this study was cut out.