Figure 3.
Activation of alternative NF-κB pathway after CD40 ligation in mature DCs. Unstimulated or LPS-treated BC1 cells were considered as immature (iDC) or mature DCs (mDC), respectively. (a) Levels of p100, RelB, or p65 in whole cell lysates. Levels of p100, RelB, or p65 in whole cell lysates of iDC or mDC were determined by immunoblotting. A representative immunoblot of three independent experiments is shown (upper). The relative intensity of the specific band is shown (lower). Each column represents the mean ± SE of three independent experiments. (b) Nuclear translocation of p100, RelB, or p65 after CD40 ligation. Cells were treated with anti-CD40 mAb (α-CD40) or control immunoglobulin for 90 min, and then the nuclear extracts were prepared. Levels of p100, RelB, p65, or histone H3 (His) in the nuclear extracts were determined by immunoblotting. A representative immunoblot of three independent experiments is shown (left). The relative intensity of the specific band is shown (right). Each column represents the mean ± SE of three independent experiments. (c) Phosphorylation of p100 after CD40 ligation. Cells were treated with anti-CD40 mAb (α-CD40) for 5–20 min, and then the whole cell lysates were prepared. Levels of phospho-p100 (pp100) or p100 in the cell lysates were determined by immunoblotting. A representative immunoblot of three independent experiments is shown (left). The relative intensity of the specific band is shown (right). Each column represents the mean ± SE of three independent experiments. Statistical significance was calculated by Student's t-test (**P < 0·01 versus unstimulated cells).