Author: Poe, Jonathan C.; Kountikov, Evgueni I.; Lykken, Jacquelyn M.; Natarajan, Abirami; Marchuk, Douglas A.; Tedder, Thomas F.
Title: EndoU is a novel regulator of AICD during peripheral B cell selection Document date: 2014_1_13
ID: 5804sjmo_49
Snippet: Gene expression analysis. Spleen B cells from mice of the indicated genotypes were cultured in triplicate for 18 h in medium alone or with F(ab) 2 anti-mouse IgM Ab, and then total RNA was isolated from pooled cells (RNeasy Plus; QIAGEN). For analysis of c-Myc and other signaling molecules, [ 32 P]-radiolabeled cDNA was generated and hybridized to GEArray NF-î«B signaling pathway gene arrays (SABiosciences) according to the manufacturer's ins.....
Document: Gene expression analysis. Spleen B cells from mice of the indicated genotypes were cultured in triplicate for 18 h in medium alone or with F(ab) 2 anti-mouse IgM Ab, and then total RNA was isolated from pooled cells (RNeasy Plus; QIAGEN). For analysis of c-Myc and other signaling molecules, [ 32 P]-radiolabeled cDNA was generated and hybridized to GEArray NF-î«B signaling pathway gene arrays (SABiosciences) according to the manufacturer's instructions, with hybridization assessed using x-ray film. Gene expression within the Chr 15 locus was analyzed using Mouse 430.2 RNA arrays (Affymetrix; Duke Cancer Inst. DNA Analysis Facility) with GeneSpring GX software (Agilent Technologies) analysis. For real-time PCR analysis, cDNA was synthesized using random primers and analyzed using a LightCycler Instrument (software version 3) and FastStart DNA Master PLUS SYBR Green I kit (Roche Diagnostics Corp), with EndoU expression quantified using EndoU exon 5 (forward) 5-CGTCAACGAGAAGCTGTTCTCCAAG-3 and EndoU exon 6 (reverse) 5-CCACATGTTCTTCAAATCGTCCAC-3 primers. Cd20 expression was the internal control. Relative EndoU expression was quantified using the REST program (version 2).
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