Fig. 3. Ser276 is a crucial residue for TNF-mediated transactivation of p65. HEK293 cells were transiently transfected with a combination of expression plasmids, i.e 180 ng of p1168hu.IL6-luc+, 20 ng of pPGKβgeobpA, 2 ng of pRcRSVp65 (or the respective mutant versions) and 100 ng of p300 expression plasmid (pCIp300 or pCIp300ΔHAT). The total amount of DNA was kept constant in all set ups by supplementing empty vector DNA. Cells were lysed 42 h after transfection. Luciferase levels were determined and corrected for transfection efficiency by normalization to β-galactosidase levels. In addition, HEK293 cells were transfected with an increasing amount of pRcRSVp65 or pRcRSVp65 S576C (30–900 ng in a total amount of 12 µg per medium Petri dish). Cytoplasmic (c) and nuclear (n) protein extracts were determined for p65 expression by western blot analysis.