Fig. 4. Sucrose density gradient analysis of TR-II in the plasma membrane of Mv1Lu cells treated with or without cholesterol and stimulated with and without TGF-1. Cells were treated with or without 50 µg/ml cholesterol at 37°C for 1 hour and further incubated with and without 50 pM TGF-1 for 2 hours. The cell lysates from these treated cells were subjected to sucrose density gradient ultracentrifugation. The sucrose gradient fractions were then analyzed by western blot analysis using anti-TR-I, anti-TR-II, anti-TfR-1 and anti-caveolin-1 antibodies. The arrow indicates the locations of TR-I, TR-II, caveolin-1 and TfR-1. Fractions 4 and 5 contained lipid rafts/caveolae whereas fractions 7 and 8 are non-lipid raft fractions. Treatment with cholesterol alone did not affect the total amounts of TGF- receptor proteins and cell proteins. Open arrowheads indicate the increased amount of TR-I or TR-II in the fraction as compared with that of untreated control. ^*The decreased amount of TR-II in the fraction as compared with that of untreated control. ^#The decreased amount of TR-II in the fraction as compared with that of treatment with cholesterol or TGF-1 alone.