Retroviral gene transduction. CD34⁺ cells or U937 cells were transduced with retroviral vectors encoding p100ΔN, empty control, RelB, or IκBα-SR upstream of IRES-GFP. (A) Schematic representation of the IκBα-SR, p100ΔN compared with wild-type (wt) p100/p52 and RelB as well as the retroviral backbone pBMN-IRES-GFP used in our study. Representative FACS diagrams show gated GFP⁺ gene–transduced cells. (B) Representative immunofluorescence analysis of RelB expression in gene-transduced DCs. Numbers indicate the percentages of the gated cells. (Upper panels) DCs transduced with RelB or empty vector. (lower panels) Subcellular localization of RelB-in RelB or p100ΔN-transduced DCs. RelB (red), GFP (green), phase-contrast and merged images. (C) Overexpression of RelB and IκBα-SR in DCs. CD34⁺ cells were cultured for 8 days and then sorted for GFP⁺ and GFP^- fractions. Lanes 1 to 4 represent total cell extracts of GFP⁺ cells transduced with p100ΔN (1), control (2), RelB (3), and IκBα-SR, as indicated. Lanes 5 to 7 represent GFP^- cells p100ΔN (1), control (2), and RelB (3). Raji cells (lane 8) were included as a RelB-positive control. (D) Western blot analysis of cytoplasmic (lanes 1-5) and nuclear (lanes 7-11) extracts prepared from FACS-sorted U937 cells transduced with the indicated constructs. Nontransduced HL60 cells (lanes 6 and 12) are compared. (E) DCs derived in vitro from CD34⁺ cells. FACS diagrams show representative nontransduced versus control-transduced cells. (F) IκBα-SR inhibits TNF-α–induced nuclear translocation of p65. Gene-transduced U937 cells were stimulated for 4 hours with 5 ng/mL TNF-α. Nuclear extracts from these cells were probed with anti-p65 and anti-SP1 antibodies (loading control). (G) Overexpression of p100ΔN was confirmed by immunoblotting of cytoplasmic U937 cell extracts with rabbit anti-p100 serum (left blot). The same extracts were analyzed in parallel for endogenous p52 levels (right blot).