Selective anchoring in the specific plasma membrane domain: a role in epithelial cell polarity

To test for cytoskeletal attachment of membrane components, we used a modification of the buffer developed by Fey et al. (25) . The monolayers were rinsed twice with extraction buffer (EB): 100 mM KC1, 2 mM MgCI2, 4 mM Na2 EGTA, 60 mM Pipes (Research Organics, Inc.), pH 6.9; and extracted for 10 min at room temperature with 0.5 % TX-100 (Sigma Chemical Co.) in EB, 1 mM PMSE We found that the addition of EGTA resulted in an excellent preservation of the membrane cytoskeleton. The cytoskeletal preparations were processed for RIA or imunofluorescence. Alternatively, they were removed in scraping buffer, treated with 0.1 mg/mi DNAase I (Sigma Chemical Co.) for 1 h at room temperature, homogenized with 50 strokes of a glass homogenizer (Dounce, Vineland, N J), exposed to various extraction conditions (see Results), centrifuged at 215,000 g for 30 min at 10°C, and processed for indirect RIA on the bottom of the ultracentrifuge tube.