Figure 4

EGFR-independent activation of NF-κB by LPA. A. LPA-induced NF-κB activation was resistant to AG1478. Caov-3 cells were starved and stimulated for the indicated periods of time with LPA (10 μM) in the presence of AG1478 (1 μM) or vehicle (DMSO). AG1478 was added 45 min before stimulation with LPA. NF-κB p65 phosphorylation at Ser 536, IκBα degradation and total IKKα/β (left panel), and IκBα phosphorylation at Ser 32 (right panel) were analyzed by immunoblotting. B. LPA-induced NF-κB activation was refractory to expression of EGFR-DN. EGFR-DN was introduced into Caov-3 cells via recombinant adenovirus (left panel) or transient transfection (right panel) and expressed as shown in Fig. 3B. LPA-induced p65 phosphorylation was analyzed by immunoblotting. C. LPA-induced NF-κB DNA-binding and transcriptional activities were unaffected by inhibition of EGFR. Nuclear extracts were prepared from Caov-3 cells stimulated for the indicated periods of time with LPA (10 μM) in the presence of AG1478 or vehicle as in A. The NF-κB DNA binding activity was determined with EMSA (left panel). LPA-induced NF-κB transcriptional activity was analyzed by luciferase assay (right panel). The cells were transfected with p5xNF-κB-Luc, starved and stimulated for 6 hours with LPA (10 μM) in the presence of AG1478 (1 μM) or vehicle. The results were presented as fold induction with the basal activity in unstimulated cells defined as 1 fold.