Fig. 3.

Modulation of occludin: occludin immunoblots of homogenates of hEVECs attached on filters. A, Estrogen-depleted (SFM) or estrogen-treated cells (Est) were co-treated for 2 d before assays with 10 μM ERα antisense oligonucleotide (ASO/ERα or Est+ASO/ERα). B, Estrogen-treated cells were cotreated for 6 h before assays with either the vehicle (Est) or lactacystin (lact., 10 μM, Est+lact). Cells were harvested, fractionated into cytosol- and plasma membrane-enriched fractions, and immunoblotted with antioccludin antibody. Similar results (not shown) were obtained with treatment with chloroquine (15 μM, 6 h). C, Cells were treated with 17β-estradiol (Est, 10 nM, 2 d); proteinase-K (Prt/k, 20 μg/ml, 30 min); or MMP-7 (10 pg/ml, plus 0.5 mM APMA, 15 min). Proteinase-K, MMP-7, and APMA were added to the luminal solution. D, Estrogen-depleted cells (SFM) were treated for 2 d before experiments with 10 μM MMP-7 antisense oligonucleotides (ASO/MMP-7) in the absence or presence of MMP-7 [10 pg/ml, plus 0.5 mM APMA, added 15 min before assays (both added to the luminal solution)]. E, Estrogen-treated cells were cotreated for 2 d before assays with the vehicle (Est) or 10 μM ERα ASO (ASO/ERα) plus MMP-7 [10 pg/ml, plus 0.5 mM APMA, added 15 min before assays (both added to the luminal solution)]. In A–E, after blotting with the antioccludin antibody, membranes were reprobed for immunoreaction with anti GAPDH antibody. The experiments were repeated three to four times with similar results.