Figure 4.

N1 and R848 synergistically activate NF-κB and MAPK signaling in human MoDCs. Serum-starved human MoDCs (2 × 10⁶ cells/ml) were treated for 0–90 min with N1 (250 ng/ml) and R848 (250 ng/ml) before they were solubilized in lysis buffer (10⁶ MoDCs/0.1 ml) to obtain whole cell lysates or in buffer (2 × 10⁶ MoDCs/0.1 ml) for analysis of the translocation of proteins from the cytoplasmic to nuclear cell fractions. An identical amount of protein was loaded and separated by electrophoresis to detect the levels of phospho-I-κBα, I-κBα, phospho-p65, phospho-p38, p38, phospho-CREB, CREB, phospho-JNK, JNK, phospho-c-Jun, c-Jun, and GAPDH in whole cell lysates (A,C) or (B) cytoplasmic fraction of phospho-p65, GAPDH and nuclear fraction of phospho-p65, lamin B1. Quantitation of band intensities from three donors using ImageJ software were normalized against GAPDH or lamin B1. **p<0.01, ***p<0.001, and ****p<0.0001 according to one-way ANOVA followed by Tukey's post hoc test.