GA inhibits TNF-induced phosphorylation and nuclear translocation of p65. (A) Immunocytochemical analysis of p65 localization. KBM-5 cells were first treated with 5 μM GA for 4 hours at 37°C and then exposed to 0.1 nM TNF for 15 minutes. After the cells were centrifuged, they underwent immunocytochemical analysis. (B,C) GA inhibits TNF-induced nuclear translocation and phosphorylation of p65. KBM-5 cells were either untreated or pretreated with 5 μM GA for 4 hours at 37°C and then treated with 0.1 nM TNF for the indicated times. Nuclear extracts were prepared and analyzed by Western blotting with antibodies against p65 and phospho-specific p65. For loading control of nuclear protein, the membrane was blotted with anti-PARP antibody.