TI - ER-alpha36 mediates testosterone-stimulated ERK activation . AB - MAPK/ERK signaling participates in the development and progression of many types of cancers including endometrial cancer [20] . To determine ER-alpha36 is involved non-genomic testosterone signaling in endometrial cancer cells , we first examined the PHOSphorylation levels of ERK , a serine-threonine kinase involved in cell proliferation [21] . As shown in Figure 2A , testosterone treatment induced PHOSphorylation of ERK1/2 in Hec1A cells . Re-probing the membrane with a total ERK1/2 antibody indicated that the total ERK1/2 content was not changed . We next examined the changes in ERK1/2 PHOSphorylation after treatment with different doses of testosterone . As shown in Figure 2B , testosterone induced a dose -dependent increase in ERK1/2 PHOSphorylation . To test the involvement of ER-alpha36 in testosterone activity observed in Hec1A cells that lack ER-alpha66 and AR expression , we decided to knockdown ER-alpha36 expression with the siRNA approach . We established a stable cell line that expresses siRNA specifically against ER-alpha36 (Hec1A/RNAi) and found that ER-alpha36 expression was down-regulated in this cell line (Figure 2C) . As shown in Figure 2D , testosterone failed to induce ERK1/2 PHOSphorylation in Hec1A/RNAi cells . Extracellular regulated kinase kinase (MEK) acts upstream of ERK1/2 to PHOSphorylate and activate ERK1/2 [22] . The MEK specific inhibitor U0126 effectively inhibited the ERK1/2 activation stimulated by testosterone (Figure 2E) . Our results indicated that the ER-alpha36 -mediated Ras/MEK/ERK pathway is involved in testosterone signaling .