Anisomycin-induced T-cell stimulatory capacity of LCs is also enhanced by nuclear RelB inhibition. (A) (Left) Empty vector controls (ctrl), d.a.MKK6-NGFR + HR-GFP (MKK6), HR-NGFR + p100ΔN-GFP (p100ΔN), or d.a.MKK6-NGFR + p100ΔN-GFP (MKK6+p100ΔN) were coinduced in immature LCs by DOX addition. CD1a⁺NGFR⁺GFP⁺ LCs were sorted, and an allo-MLR was performed. (Right) HR-GFP control vector (ctrl) or p100ΔN-GFP (p100ΔN) was induced by DOX either in the presence or absence of 40 ng/mL anisomycin. CD1a⁺GFP⁺ LCs were sorted and subjected to an allo-MLR. Diagrams represent 1 representative experiment. (B) Representative Western blot analysis for phosphorylated or total p38 MAPK of total-cell lysates from immature LCs stimulated with 40 ng/mL anisomycin for the indicated time points. The representative histogram overlay shows GFP induction (NF-κB activity) analyzed by FACS. U937 NF-κB–GFP reporter cells were left untreated or stimulated with 40 ng/mL anisomycin or 2.5 ng/mL TNFα for 24 hours. (C) Control vector (ctrl) or p100ΔN-GFP (p100ΔN) was induced in LCs either in the absence or presence of 1 μg/mL LPS or LPS + 20 ng/mL IL-1β. CD1a⁺GFP⁺ LCs were sorted, and allo-MLRs were performed. In parallel, CD1a⁺GFP⁺NGFR⁺ LCs induced to coexpress control vectors (ctrl), d.a.MKK6 + control (MKK6), MKK6 + p100ΔN, or p100ΔN + control (p100ΔN) were subjected to an allo-MLR. (D) Representative Western blot analysis of nuclear and cytosolic extracts from day 8 primary immature LCs either left unstimulated (n.s.), stimulated with 1 μg/mL LPS, or stimulated with 500 ng/mL CD40L for 48 hours. Extracts were probed with anti-RelB and antiactin antibodies.