FIGURE 8.

Phb1/2 and the CD86 cytoplasmic domain are each required for the CD86-dependent nuclear localization of NF-κB (p65). A, CH.12LX B cells were transfected via either under Mock conditions, or with scrambled negative control shRNA, or Phb1/2-specific shRNA plasmids via nucleofection for 24 hours followed by CD40L/IL-4-priming for an additional 16 hours. The cells were then resuspended in serum-free conditions for at least 30 minutes. A CD86 Ab (anti-CD86) was added to cell cultures for 90 minutes. Nuclear-enriched protein lysates were prepared and the level of p65 phosphorylation (p-p65) and total levels of p65 protein present in the nucleus were measured via immunoblot relative to Lamin A/C. B, WT, or CD86-cytoplasmic deficient (KKPΔ) FLAG-CD86 plasmids were transfected into CH12.LX B cells via nucleofection and primed with CD40L/IL-4 for 16 hours. An anti-FLAG Ab was added for 90 minutes relative to a species- and isotype-matched control Ab (iso ctrl Ab). The level of p65 phosphorylation and total levels of p65 present in the nucleus were determined via immunoblot. Densitometry was performed and measured p-p65 and total p65 band intensity present in the nucleus relative to Lamin A/C and the data are presented as the mean Fold Change in p-p65 or total p65 from primed B cells where CD86 was engaged relative to priming alone (A) or iso ctrl Ab (B) and expressed as the mean Fold Change ± SEM from three independent experiments. Statistical differences are shown relative to priming alone (A) or iso ctrl Ab (B). *, p < 0.05.