Nongenotropic regulation of cytoplasmic kinases by sex steroids. HeLa cells were transfected with reporter constructs in which SRE or AP-1 drive the expression of SEAP. Aliquots of these cells were then cotransfected with either the full-length ERα, or its ligand-binding domain (E), or the E domain fused to a membrane localization sequence (E-Mem) or nuclear localization sequence (E-Nuc) and treated with vehicle (Veh) or E2 (10–8 M) for 15 minutes. The steroid-containing media were removed, the cells were washed twice, and the cultures were continued in fresh medium without E2. Supernatants were collected 6 hours later, and SEAP activity was assayed (a). MLO-Y4 cells transfected with GFP-ERK2 and nRFP were treated for 5 minutes with 10–7 M of the indicated compounds, and nuclear accumulation of GFP-ERK2 (left upper panels of b) was quantified as described in Methods and shown in left graph. The expression of nRFP in the nuclei is shown in the left lower panels of b. Regulation of IL-6 activity by 10–8 M E2, estren, or pyrazole is shown in the right graph (b). RLU, relative luciferase units. HeLa cells transfected with the ERα (c) or the AR (d) were exposed to vehicle or 10–8 M of the indicated steroids for 15 minutes as described in a, and SEAP activity was assessed 6 hours later. One hundred percent indicates activity in vehicle-treated cells. Bars indicate means ± SD of triplicate determinations; *P < 0.05 versus vehicle by ANOVA.