Compartmentation of the Golgi complex: brefeldin-A distinguishes trans- Golgi cisternae from the trans-Golgi network

Cells were labeled with [35S]methionine and cysteine (Translabel; Amersham Corp., Arlington Heights, IL) at 0.1 mCi/ml in aMEM lacking cysteine and methionine but containing 10% dialyzed FCS. Chase periods were initiated by washing cells twice in TD (25 mM "Iris C1, pH 7.4, 5.4 mM KC1, 137 mM NaC1, 0.3 mM NazHPO4), followed by addition of complete media. After the times indicated, cells were washed once in ice-cold TD and lysed with 1.0 ml RIPA buffer (50 mM Tris HCI pH 7.2, 0.15 M NaCI, 1% Triton X-100, 1% deoxyeholate, 0,1% SDS, and 0.1% gelatin). Lysates were centrifuged at 330000 g for 10 rain in a centrifuge (model TL-100; Beckman Instruments, Fullerton, CA). Suspension cells were labeled at a density of ~2 x l(P/ml.