The PGN induced increase in W luciferase activity was inhibited by transfection of cells for 24 h with RacN17 or AktDN. As shown in Fig. 4A, activation of cells with 30 g/ml PGN induced IKK phosphorylation in a time dependent fashion. The response peaked at 30 min, started at 5 min, and declined after 60 min of treatment. The protein amount of IKK was not suffering from PGN treatment. Transfection of cells with RacN17 for 24 h, or pre-treatment of cells with LY 294002 and the Akt inhibitor for 30 min considerably attenuated PGNinduced IKK phosphorylation by 37 9%, 75 Lapatinib structure 80-day, 71 1-1.jpg, and 64 14%, respectively. Furthermore, RacN17 also inhibited the basal level of IKK phosphorylation. None of the treatments had any impact on IKK expression. Recent results suggest that phosphorylation of the p65 subunit of NF B subunits positively handles NF T transcriptional activity. To examine whether phosphorylation of the p65 adds to PGN caused NF W transactivation, we established p65 phosphorylation at Ser536 in reaction to PGN. Activation of cells with 30 g/ml PGN induced increases in p65 phosphorylation at Ser536 in a time-dependent manner. The response started at 10min, peaked at 30 min, and declined after 60 min of treatment. The protein amount of p65 was not afflicted with PGN therapy. We further examined whether p65 phosphorylation at Ser536 happened through the Rac1/PI3K/Akt signaling pathway. PGN caused p65 phosphorylation at Ser536 was significantly inhibited by transfection of cells for 24 h with RacN17 or AktDN, and by pretreatment of cells for 30 min with LY 294002. Additionally, 10 Michael LY 294002 also inhibited the basal level of p65 phosphorylation Meristem at Ser536. Nevertheless, the protein amount of p65 was not suffering from these treatments. We further examined if the activation of NF B does occur through the Rac1/PI3K/Akt signaling pathway. Being an indicator of NF B activity using transient transfection with pGL2 ELAM W luciferase, we discovered that treatment of cells with 30 g/ml PGN for 24 h caused an increase in B luciferase activity by 5. 2 0. 4 fold. or by pretreating cells for 30min with 54 7%, LY 294002, and the Akt inhibitor by ubiquitin-conjugating 45 8%, wortmannin, 33 8%, 58 91-95, and 46 7%, respectively. Taken together, these data suggest that activation of the Rac1/PI3K/Akt process is necessary for PGN caused NF B activation in RAW264. 7 macrophages. 3. 6. Rac1 is connected with TLR2 by p85 upon PGN stimulation The fast activation of Rac1 by PGN stimulation implies that Rac1 activation might occur near TLR2 in the PGN transmission pathway. For that reason, we examined whether PGN could stimulate the interaction among Rac1, p85, and TLR2. As shown in Fig. 7A, therapy of RAW 264. 7 macrophages with 30 g/ml PGN caused the association of Rac1 and TLR2, as detected by immunoblotting using the antibody to TLR2 after immunoprecipitation of Rac1.