Caveolae isolation

Caveolae isolation was performed as described previously [50, 54, 62]. Briefly, passage 3 P-UAECs were grown to approximately 90% confluence in a T75 flask and then split (passage 4) into 10 T75s for each cell line; five T75s were used as control the others for estrogen treatment. Following treatment, for each cell line, cells were collected in 0.5 mL sodium carbonate buffer (pH = 11) containing phosphatase and protease inhibitors. The proteins from the respective five T75s were pooled and then caveolar isolation was performed using discontinuous sucrose density gradient centrifugation. A light scattering band confined to the 5–35% sucrose interface was enriched with caveolar membranes. Fifteen 1-ml fractions were collected and analyzed using western immunobloting for ESR1, ESR2, and Cav-1 proteins.