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. Author manuscript; available in PMC: 2011 May 27.
Published in final edited form as: J Immunol. 2010 May 7;184(12):7207–7218. doi: 10.4049/jimmunol.0903487

Figure 3. EP4 receptor activation in BV-2 cells increases PKA activity and reduces LPS-induced phosphorylation of Akt.

Figure 3

(A) PKA activity assay of BV-2 cells stimulated with LPS (100 ng/ml), AE1-329 (100 nM), or both shows significant increases with AE1-329 and LPS+AE1-329 (*p<0.05; n=5 samples per condition). (B) Inhibition of PKA with H89 at 5 μM and 10 μM reverses AE1-329-mediated increase in PKA activity (*p<0.05 and **p<0.01). (C) Representative quantitative Western analysis of p-Akt and total Akt shows an increase in p-Akt with LPS (100 ng/ml) treatment that is reduced with stimulation with EP4 agonist AE1-329 (100 nM). BV-2 cells were treated with LPS +/- AE1-329 or vehicle and harvested at time points of 5, 15, 30, and 60 minutes; cell lysates were immunoblotted for phosphorylated Ser473 Akt (p-Akt) and total Akt. The average densitometry from three experiments is shown in the lower panel. p-Akt/Akt values have been normalized to the average signal at time=0 minutes of LPS and LPS+AE1 values. There was a significant effect of AE1-329 treatment [F(1,4)=4.589, p<0.05] and of time [F(1,4)=7.72, p<0.001]. Densitometric measurements of effects of vehicle vs AE1-329 alone did not show differences (data not shown). (D) ELISA of phospho-Thr308 Akt and total Akt at 60 minutes after stimulation with LPS +/- AE1-329 shows a significant increase in p-Akt/Akt levels with LPS stimulation, which is reversed with co-administration of 100 nM AE1-329 (*p<0.05; **p<0.01; n=6 per condition).