FIGURE 5.

Environmental pH and STAM expression control the subcellular localization of Hrs. A, cell fractionation of HEK293T, 2HP67, SL, and DL cells. Cells were homogenized at the indicated pH. Cytosolic (C) and membrane (M) fractions from post-nuclear supernatants were prepared by ultracentrifugation at 100,000 × g for 15 min at 4 °C and subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. B, ESCRT-0 proteins fractionated in the membrane fraction at pH 5.5 are not aggregates. The cell membrane prepared at pH 5.5 as shown in A was suspended in 50 mm HEPES, pH 7.2 buffer (pH 7.2 buf), or solubilized with 1% Triton X-100 (TX-100), and then separated into supernatant (S) and precipitate (P) by ultracentrifugation. The samples were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. The distribution of lysosomal integral membrane protein LAMP-1 and Triton X-100-insoluble raft-associated protein flotillin-1 were verified as the control.