ER-α36 mediates testosterone-stimulated ERK activation. Time-course analysis of ERK1/2 phosphorylation in Hec1A cells treated with 10 nM testosterone (Test) for the indicated time points. The blot was stripped and re-probed with an anti-ERK1/2 antibody (A). Hec1A cells were treated 5 min with different concentrations of testosterone, and then lysates were immunoblotted with a phospho-specific antibody of ERK1/2. The same blot was stripped and probed with an anti-ERK1/2 antibody (B). (C), Western blot analysis of ER-α36 expression in Hec1A/V and Hec1A/RNAi cells. Western blot analysis of phospho-ERK1/2 in Hec1A/V and Hec1A/RNAi ER-α36 cells treated with 10 nM testosterone for 5 min. The same blot was stripped and probed with an anti-ERK1/2 antibody (D). Lysates were prepared from Hec1A cells treated with vehicle (DMSO) (Lanes 1), 10 nM testosterone (Lanes 2 and 4) or pre-treated with 10 μM U0126 (Lanes 3 and 4) for 30 min and immunoblotted with antibodies against phospho-ERK1/2 or total ERK1/2 (E).