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Figure 6


Fig. 6. PGE2-receptor stimulation leads to activation of RhoA and inactivation of Rac1 and Cdc42. (A) Activation of the small GTPase RhoA in response to PGE2. GTP-RhoA levels were determined in lysates (pd, pull down; TL, total lysate) of HL-60 cells using a Rhotekin-GST pull-down assay. In cells treated with PGE2 for 5 minutes, Rho-GTP levels showed a 2.6±0.9 (s.d.)-fold increase relative to untreated cells (n=7). (B) Loss of Rac1 GTPase activity in response to PGE2. Rac1-GTP levels were determined in lysates of HL-60 cells using a biotinylated Pak1 peptide. In cells stimulated with PGE2, Rac1 activity was decreased 0.5±0.2 (s.d.)-fold relative to untreated cells (n=6). (C) Loss of Cdc42 GTPase activity in response to PGE2. Cdc42-GTP levels were determined in lysates of HL-60 cells using a biotinylated Pak1 peptide. In cells stimulated with PGE2, Cdc42 activity was decreased 0.7±0.2 (s.d.)-fold relative to untreated cells (n=6). (D,E) Activation of the small GTPase RhoA in response to PGE1-OH or 8-Bromo-cAMP. GTP-RhoA levels were determined in lysates of HL-60 cells using a Rhotekin-GST pull-down assay. (D) In cells treated for 5 minutes with an EP4 agonist (PGE1-OH), Rho-GTP levels showed a 1.6±0.5 (s.d.)-fold increase relative to untreated cells (n=8). (E) In cells treated for 5 minutes with 8-Bromo-cAMP (8Bromo, 200 µM), which elevates cAMP levels, Rho-GTP levels showed a 2.5±0.9 (s.d.)-fold increase relative to untreated cells (n=5). Asterisks indicate significant differences (P<0.05).





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