Effect of antioxidants and dominant-negative p67phox on TNF-induced JNK activation. (A) Time-course of JNK activation was first determined by exposing HUVECs to TNF for up to 30 minutes and probing cell lysates for phospho-JNK (p-cJun) and total JNK (total-cJun). JNK activation was apparent within 5 minutes and was maximal by 15 minutes after TNF exposure. (B) HUVEC monolayers exposed to TNF in the presence of NAC or MnTBAP were lysed and examined for JNK activity using a functional JNK assay. NAC reduced JNK activity by 60%, and MnTBAP reduced it by 90%. (C) JNK activity was also determined in HUVECs infected with either Ad-lacZ or Ad-DNp67 and exposed to vehicle or TNF. Ad-lacZ had no effect on TNF-induced JNK activity, whereas Ad-DNp67 reduced JNK activity by 55%.