, we measured the pattern of MedChemExpress Luteolin 7-glucoside intracellular vesicles in macrophages exposed to CpG-DNA, which activates the TLR9 signaling cascade. In CpG-DNA-stimulated macrophages, YB-1 secretory vesicles were clearly detected to an extent similar to LPS. Interestingly, unlike macrophages, YB-1 was distributed throughout the cytoplasm, with one or two speckles close to the nuclear membrane in LPS- or CpG-DNAstimulated bone marrow-derived dendritic cells . Therefore, YB-1 exhibits differential subcellular distribution that varies according to cell type, resulting in the regulation of distinct biological functions in macrophages and dendritic cells. To confirm these findings, we examined YB-1 secretion from macrophages and dendritic cells following LPS stimulation. Equal numbers of macrophages and dendritic cells were exposed to LPS or CpG-DNA for 24 h and then the supernatants were immunoblotted with anti-YB-1 antibody. Macrophages showed secretion of YB-1, whereas no release of cytosolic protein was observed from dendritic cells. The actual molecular weight of YB-1 is 35.9 kDa; however, this intracellular protein exhibits an electrophoretic mobility of,50 kDa. Interestingly, the molecular mass of intracellular YB-1 was detectable at 50 kDa but the majority of extracellular YB-1 exhibited the,37 kDa size along with several other molecular weight species, indicating that YB-1 may undergo fragmentation processing at different sites in LPS-stimulated macrophages. Because YB-1 knockdown resulted in no detectable protein bands in the immunoblot assay, it was confirmed that the protein bands observed were not due to nonspecific proteins. Therefore, we conclude that YB-1 is secreted in a cell type-specific manner. production but not TNF-a. To confirm these findings, we examined the effect of YB-1 depletion on IL-6 mRNA production by quantitative RT-PCR in macrophages and dendritic cells. The quantity of IL-6 mRNA produced upon LPS stimulation was increased in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19689163 YB-1-depleted macrophages and was decreased in dendritic cells under the same conditions. Similarly, RT-PCR confirmed that YB-1 affected IL-6 mRNA production in macrophages and dendritic cells in opposite ways. Therefore, we conclude that YB-1-mediated IL-6 mRNA production is dependent upon cell type and tightly coregulated with differential localization patterns of YB-1. YB-1 negatively regulates intracellular IL-6 mRNA levels in macrophages YB-1 binds near the cap structure of mRNA and also interacts with granulocyte monocyte colony stimulating PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19692147 factor mRNA. 
YB-1 consists of three major domains: a glycinerich N-terminal domain, a C-terminal domain containing alternating charged amino acids, and a highly conserved cold shock domain. The glycine-rich and cold shock domains of YB-1 are thought to bind nucleic acid, based on previous reports and our results. Therefore, we determined whether YB-1 could bind IL-6 mRNA by conducting IL-6 mRNA pull-down analysis with the indicated primers using LPS-stimulated macrophages. We demonstrated that YB-1 interacts with IL-6 mRNA but not TNF-a mRNA. Because YB-1 depletion increased IL-6 production, the ability of YB-1 to bind IL-6 mRNA suggested a possible role of YB-1 in regulating IL-6 mRNA stability. To investigate this possibility, we treated LPS-stimulated macrophages with the transcriptional inhibitor actinomycin D and then examined IL-6 mRNA stability by RT-PCR. We found that the amount of IL-6 mRNA during ActD exposure was much higher than in control macrophage