PI3K-dependent activation of NF-κB by P. gingivalis. (A) Activation of NF-κB-dependent luciferase in HEK cells. Monolayers were infected with wild type P. gingivalis in the presence of no drug (Open bars), 20 μM SN50 (Speckled bars), 20 μM LY294002 (Striped bar), or with the triple gingipain mutant (Closed bars), and luciferase assay carried out as described in Materials and Methods. Error bars represent average of three experiments. (B) Nuclear import of NF-κB p65 subunit. HGF cells were infected with P. gingivalis (wild type = WT; gingipain mutant = MT), and nuclear extracts (40 μg protein) of infected cells (or uninfected controls = U) were analyzed for p65 by immunoblot . Sp1 serves as an unchanged control. (C) Phosphorylated IκB-α, an indicator of NF-κB activation, was detected by immunoblot of the total infected HGF cell extract using a specific antibody . This phosphorylation was undetectable when 20 μM LY294002 (PI3K inhibitor) was included in the culture medium (+I).