Figure 6

St-HT31 abolished the cAMP inhibition of PMA-induced ERK1/2 activation.

(A) St-HT31 abolished the cAMP inhibition of PMA-induced ERK2 activation in human spermatozoa. Human spermatozoa were washed and preincubated with St-HT31 or the inactive peptide inhibitor St-HT31-P (both 20 μM) for 20 min followed by another preincubation with or without 8-Br-cAMP (1 mM) for 10 min. Thereafter PMA (25 nM) was added for another 15 min. cell lysates were analyzed for ERK2 activity by western blotting as above. A representative blot is shown and bars are mean ± SEM from three experiments. (B) St-HT31 abolished the cAMP inhibition of PMA-induced ERK1/2 activation in HEK293T cells expressing proAKAP4. HEK293T cells were transfected with turboGFP-proAKAP4. Following the transfection (48 hours) cells were pretreated with St-HT31, or St-HT31-P (20 μM) for 20 min, and were then preincubated with or without 8-Br-cAMP (1 mM) for 10 min. Thereafter PMA (25 nM) was added for another 60 min. Cell lysates were analyzed for ERK2 activity by Western blotting as above. A representative blot is shown and bars are mean ± SEM. from three experiments. *(p < 0.05) **(p < 0.01).