Figure 5

Indirect role of v-FLIP-induced cytokines in the induction of various signaling molecules. (a) Cell lysates prepared from serum-starved (8 h) HMVECs incubated with supernatants collected from serum-starved (8 h) p-SIN and v-FLIP/K13-HMVECs cells for 4 h, 8 h and 24 h. (b) Cell lysates were western blotted using p-FAK, p-Src, p-AKT, p-P38, p-ERK and p-RSK antibodies. These blots were stripped and blotted with total antibodies against FAK, Src, AKT, P38, ERK and RSK. (c) Cell lysates prepared from serum-starved (8 h) HMVECs incubated with supernatants collected from untreated serum-starved (8 h) p-SIN, untreated v-FLIP/K13-HMVECs and v-FLIP/K13-HMVECs pretreated with either NS-398 or celecoxib for 8 h. These supernatants were used to induce signaling in serum-starved (8 h) HMVECs for 4 h for FAK, Src, ERK, P-38 and RSK, and 8 h for AKT. (d, e) Rac1-GTPase activity in the lysates prepared in (b) and (c). Histogram representing Rac1-GTPase activation as measured by G-LISA. Fold activation of Rac1 is calculated by considering Rac1-GTPase activity in the p-SIN-HMVECs as onefold. Each reaction was done in triplicate, and each bar represents the mean±s.d. for three experiments. ^* and ^**: statistically significant at P<0.01 and P<0.005, respectively. The % Inhibition in invasion by NS-398 or celecoxib treatment was calculated considering invasion of untreated v-FLIP/K13-HMVECs as 100%.