Impaired NF-κB activity in Bcl-3-transduced and IL-10-pretreated macrophages. (A) Western blot showing the nuclear expression of p65 subunit of NF-κB after LPS stimulation (1 hour) in RAW cells transduced with GFP alone or Bcl-3/GFP. (B) RAW cells transduced with GFP alone or Bcl-3/GFP were stimulated with LPS and nuclear extracts were subjected to EMSA. There are 2 types of NF-κB binding to the probe, p50/p65 heterodimers and p50/p50 homodimers, indicated by the arrow and arrowhead, respectively. (C) Western blot showing the nuclear expression of p65 before and 60 minutes after LPS stimulation in nonpretreated (med) and IL-10-pretreated (IL-10) RAW macrophages. (D) RAW cells were pretreated with 10 ng/mL IL-10 for 18 hours and stimulated with 10 ng/mL LPS for 60 minutes. Nuclear extracts were subjected to EMSA using the NF-κB binding site of the TNF-α promoter as a probe. The specificities of the shifted bands were determined by adding specific Abs to p50 (arrow) and p65 (arrowhead). Similar results were observed in mouse peritoneal macrophages.