Figure 4.

Efficient TNF‐induced degradation of IκBα and concomitant liberation of RelA requires functional p97/VCP. Cells stimulated with TNF (10 ng/ml) for the indicated times after either pretreatment with p97/VCP inhibitor NMS‐873 (2.5 μM, NMS ³) for 10 min. or inhibitor treatment (2.5 μM) from 15 min. after stimulation in either the presence (NMS ¹) or the absence (NMS ²) of pan‐caspase inhibitor ZVAD‐fmk (10 μM), were subjected to subcellular fractionation. LMB (10 ng/ml) was applied 15 min. after stimulation to prevent Crm1‐dependent nuclear export of RelA. Samples were analysed by IB by use of indicated antibodies. Tubulin (cytosol), Nucleolin (N1) or Lamin B2 and HDAC1 (N2) were used as marker proteins for the respective subcellular fractions and detected for control of equal protein load. Detection of γH2AX on one hand and caspase and PARP1 cleavage on the other was accomplished to determine induction of DNA damage and or apoptosis respectively.