8A and B). These results suggest that the more activated STATs existed, more the interacting partners were retained in the cytoplasm, which in turn, probably through the increased STAT complex formation, leads to the promotion of antagonistic actions by IFN-α and IL-4 in Ramos B cells. Likewise, the STAT2 knock-down experiments indicated that lack of STAT2 prevented IFN-α-induced cytosolic retention of IL-4-activated pY-STAT6, and almost abrogated IL-4-mediated PI3K Inhibitor Library order inhibition of IFN-α

action on the IRF7 induction. The results support that the formation of STAT6:STAT2 complex is playing a critical role in cross-suppression of IL-4 and IFN-α signal transduction and the resulting biological response (Supporting Information Fig. S6). In summary, our data obtained in a B-cell system demonstrate that antagonism by IL-4 and IFN-α is mediated by a novel two-way signal cross-talk mechanism, involving the molecular complex formation and cytoplasmic retention of IL-4-induced pY-STAT6 and IFN-α-induced pY-STAT2:p48. The subsequent Selleck GPCR Compound Library attenuation of nuclear localization of the phosphorylated STATs and reduced transcription

by respective STATs would then be responsible for the counter-regulation of biological responses by these cytokines. The human Ramos B (RA1) cells were maintained as described 40. PBMCs were isolated using Ficoll-hypaque from the blood obtained from healthy donors. Human recombinant IL-4 was obtained from R&D systems (Minneapolis, MN, USA). Human recombinant IFN-α and IFN-γ were obtained from LG Life Sciences (Daejeon, Korea) and R&D systems. Stimulation of cells with IL-4, IFN-α, or IFN-γ was done in 0.1% FBS-containing RPMI media. Gene transfection by electroporation was performed as described 41. pCS3-MT-STAT2-myc and pXM-STAT6 vectors were provided by Dr. J. H. Ahn (Sungkyunkwan University, Korea) and Dr. B. Groner (Johann Wolfgang Goethe University, Germany), respectively. Cell surface expression of CD23 was N-acetylglucosamine-1-phosphate transferase analyzed by FACSCalibur (BD Bioscience, San Diego, CA, USA), using PE-conjugated

antihuman CD23 mAb (BD Bioscience). The levels of CD23 were expressed as arithmetic ΔMFI, which was calculated by subtracting MFI of the samples stained with an isotype-matched negative control antibody from that of the samples stained with specific antibodies. Total RNA was isolated with Trizol reagent (Invitrogen, Camarillo, CA, USA)/Ribospin™ (GeneAll Biotechnology, Seoul, Korea) and then reverse-transcribed, after which real-time PCR amplification with iQ SYBR Green (Bio-Rad, Hercules, CA, USA) was performed using a Mastercycler realplex thermalcylcer (Eppendorf AG, Hamburg, Germany), using the primers shown below. human CD23, 5′primer : gtcccaggaattgaacgaga, 3′primer : ccatgtcgtcacaggcatac, human IRF7, 5′primer : taccatctacctgggcttcg, 3′primer : gctccataaggaagcactcg, human GAPDH, 5′primer : gacatcaagaaggtggtgaa, 3′primer : tgtcataccaggaaatgagc.