Figure 6

LMP1 increased the binding ability of c-Jun and c-Fos transcription factors to κAP-1 motif in vitro. (A) Biotin-labeled wild-type κAP-1 oligonucleotide probe was incubated with nuclear extracts of HNE2, HNE2-LMP1, HNE2-LMP1-TAM67 and SP600125-treated HNE2-LMP1 (20 μM for 12 hr) NPC cells in the presence of a 200-fold excess of unlabeled wild-type κAP-1 (lane 6), unlabeled mutant κAP-1 oligonucleotides (designated as mutAP-1, lane 7) or noncompetitive unlabeled κNF-κB oligonucleotide (NS, lane 8), and then AP-1 DNA binding activities were examined by EMSA. (B) Biotin-labeled mutant κAP-1 oligonucleotide probe was incubated with nuclear extracts of indicated the NPC cell lines, and then AP-1 DNA binding activities were examined by EMSA. (C) 10 μg of HNE2-LMP1 nuclear extracts were preincubated with biotin-labeled κAP-1 oligonucleotide probe in the absence (lane 2) or presence of antisera directed against c-Jun (lane 3), c-Fos (lane 4) and then supershift assays were performed. (D) Phosphorylation levels of JNK and c-Jun in HNE2 and HNE2-LMP1 cells. The nuclear extracts were prepared and analyzed with phospho-JNK (Thr183/Tyr185), phospho-c-Jun (Ser63) and phospho-c-Jun (Ser73) antibodies, respectively. Nucleolin was acted as a nuclear protein loading control. (E) Expression of c-Jun and c-Fos in HNE2 and HNE2-LMP1 cells. Whole cell lysates were prepared and expression of c-Jun and c-Fos was estimated by Western blotting. α-tubulin was detected as a protein loading control.