Project description:The activation signaling of transcription factor nuclear factor-kB (NF-kB) plays central role for immune system. One of key kinase mediating this pathway is TAK1 in adaptive and innate immunity. However, role of TAK1 in B cell receptor signaling is still unclear. To know effects of TAK1-deletion on the gene expression induced by anti-IgM, we performed the time course analysis in comparison of wild type with TAK1-deleted splenic B cells. Splenic B cells were purified by depleting CD43+ cells. Purified B Cells were stimulated with 10 µg/ml of anti-IgM for 1, 3, 6, or 24hr. Two replicated samples were analysed. Unstimulated cells (0) were control.
Project description:The activation signaling of transcription factor nuclear factor-kB (NF-kB) plays central role for immune system. One of key kinase mediating this pathway is TAK1 in adaptive and innate immunity. However, role of TAK1 in B cell receptor signaling is still unclear. To know effects of TAK1-deletion on the gene expression induced by anti-IgM, we performed the time course analysis in comparison of wild type with TAK1-deleted splenic B cells.
Project description:BCR-induced gene expression profile in wild-type and B cell-specific TAK1-deficient B cells; to elucidate how TAK1 regulates BCR-mediated proliferative response Keywords: ordered
Project description:BCR-induced gene expression profile in wild-type and B cell-specific TAK1-deficient B cells; to elucidate how TAK1 regulates BCR-mediated proliferative response Experiment Overall Design: Purified splenic B cells (CD43 negative) were treated with or without anti-IgM (20 micloG/mL) for 4 h. Total RNA was extracted with an RNeasy kit (Qiagen), double-stranded DNA was synthesized from 10 micloG of total RNA with the SuperScript Choice System (Invitrogen) primed with T7-(dT) 24 primer. These cDNAs were used to prepare biotin-labeled cRNA by an in vitro transcription reaction performed using T7 RNA polymerase in the presence of biotinylated-ribonucleotides, according to the manufacturerâs protocol (Enzo Diagnostics). The cRNA product was purified using an RNeasy kit, fragmented, and hybridized to Affymetrix mouse expression array A430.2 microarray chips, according to the manufacturerâs protocol (Affymetrix). The hybridized chips were stained, washed, and scanned with a GeneArray Scanner (Affymetrix).
