Fig. 4.

Activation of ERK following estradiol (E₂) or 4,4′,4″-(4-propyl-[1H]pyrazole-1,3,5-triyl)tris-phenol (PPT) treatment in primary male and female vascular smooth muscle cells (VSMCs). I,A: representative photomicrographs of aortic VSMCs (a and b). Scale bar = 200 μm. Fluorescent images of immunoreactive VSMCs against α-smooth muscle actin antibody (c and d, green) are shown. Cells were visualized by nuclear DAPI staining (blue). Scale bar = 50 μm. I,B: representative Western blots for the levels of phosphorylated (p)ERK1/2 and total ERK1/2 after E₂ (1 μM) stimulation of male and female VSMCs. β-Tubulin is shown as a loading control. I,C: quantitation of I,B. The level of pERK1/2 was normalized to that of total ERK1/2 (n = 3 experiments, #P < 0.05 and ##P < 0.01, E₂ treatment vs. vehicle controls). I,D: representative Western blots for the levels of pERK1/2 and total ERK1/2 after PPT (1 µM) stimulation of VSMCs. I,E: quantitation of I,D. pERK1/2 expression was normalized to that of total ERK1/2. Data were collected from 3 independent experiments where primary VSMCs were generated for each experiment (n = 3 experiments, #P < 0.05 and ##P < 0.01, PPT treatment vs. vehicle controls). II,A: effect of the ERK inhibitor PD-98059 on expression of pERK following E₂ or PPT stimulation in primary male and female mouse VSMCs. Representative Western blots are shown for levels of pERK1/2 and total ERK1/2 after E₂ (1 µM) or PPT (1 µM) stimulation for 10 min with or without PD-98059 (10 µM) preincubation for 30 min. II,B: effect of the ERK inhibitor PD-98059 on the expression of pERK after E₂ or PPT stimulation in primary male and female mouse VSMCs (quantitation of II,A). The level of pERK1/2 was normalized to that of total ERK1/2 (n = 3 experiments, #P < 0.05 and ##P < 0.01). Data are expressed as means ± SE.