Figure 4

MA blocks TNFα-induced p65 nuclear translocation, p65 phosphorylation and IκBα degradation in pancreatic cancer cells. (A) MA inhibited TNFα-induced p65 nuclear translocation by immunofluorescence analysis. Panc-28 cells were pretreated with or without 25 microM MA, and then stimulated with or without 0.1 nM TNFα for 20 minutes. The localization of p65 was visualized by immunofluorescence analysis as described in materials and methods. Magnification, ×40. (B) MA inhibited TNFα-induced p65 nuclear translocation and p65 phosphorylation by Western blot. Panc-28 cells were incubated with 25 microM MA for 6 h and treated with 0.1 nM TNFα for the indicated time. Cytoplasmic extracts (CE) and nuclear extracts (NE) were analyzed by Western blot using the indicated antibodies. Anti-β-actin and anti-PARP antibodies were used as controls. (C) MA inhibited TNFα-induced IκBα degradation. Cells were pretreated with 25 microM MA, and then stimulated with 0.1 nM TNFα for 20 minutes. Whole cell extracts were prepared for Western blot analysis using indicated antibodies.