Figure 4

LMP1 increased the binding ability of p52 and p65 transcription factors to κNF-κB motif in vitro. (A) Biotin-labeled wild-type κNF-κB oligonucleotide probe was incubated with nuclear extracts of HNE2, HNE2-LMP1, HNE2-LMP1-DNMIκBα and Bay11-7082-treated HNE2-LMP1 (20 μM for 12 hr) NPC cells in the presence of a 200-fold excess of unlabeled wild-type κNF-κB (lane 6), unlabeled mutant κNF-κB oligonucleotides (designated as mutκB, lane 7) or noncompetitive unlabeled κAP-1 oligonucleotide (NS, lane 8), and then NF-κB DNA binding activities were examined by EMSA. (B) Biotin-labeled mutant κNF-κB oligonucleotide probe was incubated with nuclear extracts of indicated NPC cell lines, and then NF-κB DNA binding activities were examined by EMSA. (C) 10 μg of HNE2-LMP1 nuclear extracts were preincubated with biotin-labeled κNF-κB oligonucleotide probe in the absence (lane 2) or presence of antibodies directed against different NF-κB subunits p50, p52, p65, c-Rel, RelB or control antibody (IgG) (indicated above each lane) and then supershift assays were performed. (D, E) Expression and subcelluar distribution of p65 in HNE2 and HNE2-LMP1 cells. Whole cell lysates, cytoplasmic and nuclear fractions were prepared and expression of p65 was estimated by western blotting. Cytosolic protein α-tubulin and nuclear protein nucleolin were detected as protein loading controls. (F, G) Expression and subcelluar distribution of p52 in HNE2 and HNE2-LMP1 cells. Whole cell lysates, cytoplasmic and nuclear fractions were prepared and expression of p52 was estimated by western blotting. Cytosolic protein α-tubulin and nuclear protein nucleolin were detected as protein loading controls.