The Effect of EBNA 2 and 3 proteins on <i>ITGAL</i> expression.

<p>(A) Q-PCR analysis of <i>ITGAL</i> transcript levels using cDNA from wild-type LCLs infected with B95.8 virus (wt) and EBNA 3B knock-out LCLs (KO) in two different donor backgrounds (PER142 and PER253). Transcript levels were normalised to GAPDH levels and expressed relative to the level in wt cells for each donor. Results show the mean −/+ range of two independent QPCR reactions each performed in duplicate. (B) Luciferase reporter assays carried out in DG75 cells transiently transfected with 2 µg of the control vector pGL3 basic, an <i>ITGAL</i> promoter-luciferase reporter (pGL3 <i>ITGAL</i>p) or the EBV C promoter reporter (pCp1425GL2) (right panel) in the absence or presence of 10 or 20 µg of EBNA 2, 3A, 3B or 3C expressing constructs. Firefly luciferase signals were normalised to Renilla luciferase signals from the co-transfected control plasmid pRL-TK (1 µg). Results show the mean −/+ standard deviation of 3 independent experiments and are expressed relative to the pGL3 basic signal (left panel) or the Cp1425GL2 signal (right panel) in the absence of EBNA 2. Western blot analysis of EBNA 2, 3A, 3B and 3C expression levels in transfected cells. Each set of blots was also probed for actin as a loading control. (C) Re-ChIP analysis in Mutu III cells using anti-EBNA 2 antibodies in the first round of ChIP followed by a second round of ChIP in absence of antibody or using anti-EBNA 2, EBNA 3B or EBNA 3C antibodies. Primers at peak 3 were used for analysis. Results show mean percentage primary input −/+ range of two independent Q-PCR reactions from a representative experiment. (D) Control re-ChIP analysis using anti-EBNA 3B antibodies in the first round followed by re-precipitation in the absence of antibody or using anti-EBNA 3B antibodies. (E) Control re-ChIP analysis using anti-EBNA 3C antibodies in the first round followed by re-precipitation in the absence of antibody or using anti-EBNA 3C antibodies.