Figure 2

Role of v-FLIP/K13 transduction-induced MAPKs in COX-2 gene expression, protein levels and PGE2 secretion. (a) Phospho-MAPK array blots for the lysates prepared from p-SIN and v-FLIP/K13 HMVECs. (b) Densitometric analysis of MAPK array blots measuring the activation of MAPKs. The values were normalized to identical background levels using the R & D Systems (Minneapolis, MN, USA) Human MAPK antibody array analysis tool. The fold induction of MAPK was calculated by dividing the respective values obtained from v-FLIP/K13-HMVEC lysates with the values obtained from p-SIN-HMVEC lysates. (c) Cell lysates prepared from p-SIN and v-FLIP/K13-HMVECs were immunoblotted using p-ERK, p-AKT1, p-P65, p-RSK1 and p-GSK3-β antibodies. These blots were stripped and blotted against total antibodies for ERK, AKT, P65, RSK1, GSK3-β and tubulin to ensure equal protein loading. (d) v-FLIP/K13-HMVECs were treated with noncytotoxic concentrations of various MAPKs inhibitors for 1 h at 37°C. Total RNA was prepared from these cells and COX-2 gene expression was quantitated as described before. The COX-2 level in uninfected cells was considered as onefold for comparison. Percentage inhibition was calculated by considering COX-2 gene expression in inhibitor-untreated, v-FLIP/K13-transfected cells as 100%. (e) HMVEC-d cells were treated as described in (d). Total protein was prepared from these cells and COX-2/COX-1 protein levels were quantitated by western blotting. % Inhibition was calculated by considering the COX-2 protein level in inhibitor-untreated, v-FLIP/K13-transfected cells as 100%. β-Actin levels were checked as loading control. (f) Supernatants collected from the HMVECs treated in (d) were used for PGE2 measurement by ELISA as described before. Data are expressed as mean pg/ml. Each reaction was done in triplicate and each point represents the average±s.d. of five experiments. % Inhibition was calculated by considering PGE2 secretion from inhibitor-untreated KSHV-infected (2 h) cells as 100%. ^* and ^**: statistically significant at P<0.01 and P<0.005, respectively.