Fig. 5.

TRAF2 regulates mouse Icam-1 gene expression upon CD40L stimulation in Neuro2a cells. a Schematic diagram of mouse Icam-1 gene promoter. b Time course of TRAF2, pRNAPolII, and p65 recruitment to mouse icam-1 promoter upon CD40L treatment in Neuro2a cells. qPCR analysis of TRAF2, p65, TRAF3, and pRNAPolII chromatin immunoprecipitation (ChIP) after 5, 10, and 30 min of CD40L (100 ng/mL) treatment. ChIP qPCR analysis shows significant increase in TRAF2 and pRNAPolII recruitment to p65 consensus sequence (p65 C S) starting 5 min after CD40L treatment. At 10 min, p65 joined TRAF2 and pRNAPolII at the promoter. Following 30 min, TRAF3 joined the complex and maximal levels of TRAF2, pRNAPolII and p65 occurred at this time. Knockdown of TRAF2 by SiRNA (SiTRAF2) abrogated p65, and pRNAPolII to the complex on the Icam-1 promoter. The TRAF3 recruitment to the promoter was not investigated since it did not show any changes in levels at the tested 10-min time point. Nonspecific IgG was used as an internal control while primers flanking an irrelevant control sequence were used as external controls. c Monoubiquitination of H2B and TRAF2 recruitment to the Icam-1 gene promoter. Left panel: qPCR analysis of TRAF2 ChIP after 10 min of CD40L (100 ng/mL) treatment show 3.7-fold increase in TRAF2 recruitment to p65 consensus sequence (p65 C S) as compared to nontreated cells. This increase is abrogated when TRAF2 was knocked down using SiRNA. Right panel: qPCR analysis of H2B-Ub (Lys 120) ChIP after 10 min of CD40L (100 ng/mL) treatment show 29.8-fold increase in H2B monoubiquitination at the p65 consensus sequence (p65 C S) as compared to nontreated cells. This increase is abrogated when TRAF2 was knocked down using SiRNA. Nonspecific IgG was used as an internal control while primers flanking an irrelevant control sequence were used as external controls. d Mouse Icam-1 mRNA expression levels upon CD40L treatment in Neuro2a cells. qPCR analysis of Icam-1 mRNA expression in Neuro2a cells treated with Scrambled, TRAF2, and p65 SiRNA at 0, 5, 10, and 30 min after CD40L (100 ng/mL) treatment. TRAF2 knockdown shows dramatic decrease in Icam-1 mRNA expression starting 5 min while p65 knockdown significantly affects Icam-1 expression at the 30-min time point. TSS transcriptional start site. Ctrl irrelevant control sequence, F and R forward and reverse primers, respectively. *p < 0.05