Fig. 3. Role of Ser311 in NF-κB transcription. (A) Subconfluent cultures of 293 cells were transfected with either empty plasmid (striped bar) or different amounts of expression vectors for HA-tagged wild-type or S311A mutant RelA along with a κB-dependent reporter and the Renilla control plasmid. Luciferase activity was determined as described in Materials and methods. Results are the mean ± SD of three independent experiments with incubations in duplicate (left panel). The expression levels of wild-type and mutant RelA of one representative experiment were determined by immunoblotting with an anti-HA antibody (right panel). (B) EFs transduced with empty pBabe vector (control) or stably expressing wild-type or S311A mutant RelA were transfected with the κB-luciferase reporter vector along with the control Renilla plasmid, after which they were stimulated for 6 h with different concentrations of TNF-α (upper left panel) or the agonistic anti-lymphotoxin-β receptor antibody ACH6 (lower panel). Luciferase activity was determined as above. Expression levels of endogenous and ectopically expressed RelA proteins were determined by immunoblotting with an anti-RelA antibody (upper right panel).