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. 2017 Oct 17;7(19):4671–4688. doi: 10.7150/thno.21216

Figure 7.

Figure 7

FSHR coupled to GαS /AC/cAMP/PKA cascade to activate PI3K/Akt/mTOR. (A). HUVECs were treated with vehicle or FSH (50 mIU/mL) for 24 h in the presence or absence of GαS antagonist NF499 (10 μM) and analyzed by Western blotting. (B). HUVECs were treated with FSH (50 mIU/mL) for over 6 h. Cell lysates were subjected to immunoprecipitation using anti-active GαS monoclonal antibody. Immunoprecipitates were analyzed by Western blotting with anti- GαS antibody. (C). HUVECs were treated with vehicle, FSH, or adenylyl cyclase activator Forskolin (FSK, 10 μM) in the presence or absence of GαS antagonist NF499. cAMP concentration was determined by ELISA. ** = P < 0.01, *** = P < 0.001 vs. control; ## = P<0.01, ### = P<0.001 vs. FSH or FSK. (D). HUVECs were treated with vehicle or FSH in the presence or absence of NF499 or adenylate cyclase inhibitor MDL12330A (MDL, 10 μM). PKA activity was determined by ELISA. ** = P < 0.01 vs. control; ## = P<0.01 vs. FSH. (E). Western blots of HUVECs treated with vehicle, FSH or FSK in the presence or absence of PKA inhibitor H89 (10 μM) pretreatment. (F). HUVECs were transfected with scrambled siRNA (100 nM) or PKA Cα siRNA (100 nM) for 48 h. Then cells were treated with vehicle or FSH (50 mIU/mL) for another 24 h and analyzed by Western blotting. All experiments were repeated at least three times with consistent results and the representative images are shown.