Figure 4

(A) Effect of COX-2 inhibition on detachment-induced cytoskeletal rearrangement-linked cellular survival kinases. (a) p65 immunostaining in untreated, NS-398- or celecoxib-treated v-FLIP-HMVECs. (b) EMSA for nuclear extracts prepared from untreated, NS-398- or celecoxib-treated v-FLIP-HMVECs using the NF-κB probe. This EMSA experiment shows the binding of nuclear extracts prepared from untreated, NS-398- or celecoxib-treated v-FLIP-HMVECs to the NF-κB probe. The specificity of the DNA–protein interaction was assessed by competition using a 100-fold molar excess of unlabeled double-stranded oligonucleotide NF-κB probe as a competitor. Each EMSA is representative of at least three independent experiments. (c) Cell lysates prepared from untreated, NS-398- or celecoxib-treated v-FLIP-HMVECs were western blotted using p-FAK, p-Src, p-AKT, p-P65, p-ERK and laminin-γ antibodies. These blots were stripped and blotted against total antibodies for FAK, Src, AKT, P65, ERK and ß-actin to confirm equal protein loading. (Ba) Representative pictures depicting p-FAK, p-Src, p-FAK/p-Src and focal adhesions. (b) Table representing the number of cells with p-FAK, p-Src or p-FAK/p-Src staining per 10 cells. (C) Laminin-γ staining of untreated or NS-398-treated v-FLIP-HMVECs by immunofluoresence microscopy. White arrows in B and C denote FAK-Y397, p-Src, FAK-Y397/p-Src and Laminin-gamma. (D) 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. ^*statistically significant at P<0.01.