LPS treatment leads to increased p65-directed miR-155 expression. (A) BJAB cells were treated with 5 μg/ml LPS for 0, 15, 30, and 60 min and nuclear extracts were made. Western blots were performed on p65, PARP (nuclear control), and β-tubulin (cytosolic control). Whole cell extracts (WCE) of BJAB cells were used as a control. (B) BJAB cells were treated with 5 μg/ml LPS as in (A) and cDNA preps were prepared. The expression levels of the unspliced MIR155HG transcript were assessed by qPCR and normalized to GAPDH. Values were normalized to untreated cells (1.0), +/- SE and * indicates p < 0.003. (C) BJAB and IB4 cells were treated with 5 μg/ml LPS for 24 h and cDNA preps for small RNAs were prepared. The mature miR-155 expression levels were then assessed by qPCR and normalized to 5S rRNA. Values were normalized to untreated samples for each cell type and the relative expression (Rel. Exp.) is relative to untreated BJAB cells. The * indicates p < 0.003. (D) BJAB cells were treated with 5 μg/ml LPS for 0 or 60 min and nuclear extracts were made. An EMSA was performed using a 32P-labeled probe containing the predicted -178 NF-κB site in the MIR155HG promoter (described in Figures 2 and 3). Where indicated, an antibody specific for p65 was added. A light exposure shows p50/p65 heterodimer binding just above the p50 homodimer band, and the darker exposure shows the supershift with p65 antibody. The last lane is a size control made from a whole cell extract of A293 cells transfected with p65 and p50 plasmids (refer to Figure 3). Nuclear extracts were also subjected to anti-p65 Western blotting (bottom panel).