FIG. 5.

EMSA detection of KSHV-induced NF-κB DNA binding activity. (A and B, top, lanes 1 to 7) HMVEC-d cells (A) and HFF (B), untreated or pretreated with 10 μM Bay11-7082 (1 h), were left uninfected or infected with KSHV (10 DNA copies/cell) for the indicated times. Nuclear extracts prepared from these cells were incubated with κB-specific probe. The specificity of DNA-protein interaction was assessed by induction with TNF-α (20 ng/ml) (lane 8), free probe (lane 9), normal probe (lane 11), or competitive EMSA using a 100× molar excess of unlabeled double-stranded oligonucleotide κB probe (cold probe; lane 10). Nuclear extracts from HMVEC-d cells infected with 10 DNA copies/cell of live KSHV (C) or UV-KSHV (D) for 2 h, 8 h, and 24 h were analyzed by EMSA. Each EMSA is representative of at least three independent experiments. (A and B, bottom, lanes 1 to 11, and C and D, bottom) Oct1 probe used as loading control and specificity control to demonstrate the binding of NF-κB to the specific probe. The arrows indicate the positions of the induced NF-κB complexes.