ER-α36 mediates testosterone-stimulated Akt activation. Western blot analysis of phospho-Akt in Hec1A cells treated with 10 nM testosterone (Test) for the indicated time points. The same blot was stripped and probed with an anti-total Akt antibody (A). Hec1A cells were treated 5 min with different concentrations of testosterone, and cell lysates were immunoblotted with a specific antibody against phospho-Akt. The same blot was stripped and probed with an anti-total Akt antibody (B). Western blot analyzed phospho-Akt in Hec1A/V and Hec1A/RNAi ER36 cells treated with 10 nM testosterone for 5 min. The same blot was stripped and probed with an anti-Akt antibody (C). Lysates were prepared from Hec1A cells treated with vehicle (DMSO) (Lanes 1), 10 nM testosterone (Lanes 2 and 4) or pre-treated with 50 μM PI3K inhibitor LY294002 (Lanes 3 and 4) for 1 h and immunoblotted with antibodies against phospho-Akt or total Akt (D).