Modulation of the effect of TTP plasma on c-FLIP through proteasome inhibition and alteration of c-FLIP ubiquitination. (A) Effect of MG132 on total FLIP expression in MVECs. Top panels show representative immunoblots. Pulmonary and dermal MVECs were incubated with normal (lane 1) or TTP plasma (lane 2; 2% vol/vol) for 30 minutes. Cultures treated with MG132 (5 μM) were exposed to this proteasome inhibitor for 1 hour prior to addition of normal (lane 3) or TTP (lane 4) plasmas. Bottom panel shows summary of 3 experiments using 2 normal and 2 TTP plasmas. MG132 had no effect on FLIP expression in pulmonary MVECs (P = .21), but it blocked TTP plasma–mediated FLIP suppression by TTP plasma in dermal MVECs (P = .048). (B) Effect of TTP plasma and cytokines on FLIP ubiquitination. Dermal MVECs were immunoprecipitated with an anti-FLIP polyclonal Ab. Equal amounts of protein (100 μg) were then loaded on gels and immunoblotted with an antiubiquitin mAb. A representative immunobot and a summary of 3 experiments are presented. Levels of monoubiquitinated FLIP (arrow; just above the dense heavy chain IgG band) were increased by TTP plasma (P = .008) and TRAIL plus IFN-γ (P = .03) compared with cells incubated with normal plasma. Lane 1, control plasma; lane 2, TTP plasma; lane 3, TRAIL plus IFN-γ. Standard deviations for all mean values are provided.