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. Author manuscript; available in PMC: 2018 Sep 5.
Published in final edited form as: J Cell Biochem. 2016 Dec 13;118(6):1387–1400. doi: 10.1002/jcb.25797

Fig. 6.

Fig. 6.

Effects of the treatment (at 37°C for 4 h) with several concentrations of 7-DHC and cholesterol on the stability of TβR-I and TβR-II in Mv1Lu cells (A) and with 50 μg/ml of 7-DHC and cholesterol on the stability of TβR-I and TβR-II localized in plasma-membrane microdomains of Mv1Lu cells (B). (A) Mv1Lu cells were treated with several concentrations (0, 5, 10, 20, 40, and 50 μg/ml) of 7-DHC (a) or cholesterol (b) at 37°C for 4 h. The cell lysates of treated cells were subjected to 7.5% SDS–PAGE, followed by Western blot analysis using antibodies to TβR-I, TβR-II, and b-actin as described previously [Huang et al., 2016a,b]. The arrow indicates the location of TβR-I, TβR-II, or b-actin. The data is representative of three independent experiments. The levels of TβR-I, TβR-II, and b-actin were quantified by densitometry. The relative levels of TβR-I and TβR-II in treated cells were estimated by normalizing the level of b-actin to that in control cells treated with vehicle only. 7-DHC at 50 μg/ml appeared to attenuate the expression of TβRI or TβR-II by ~30% (marked by an open arrowhead) as compared to that in control cells treated with vehicle only. (B) Cells were treated with cholesterol (50 μg/ml) or 7-DHC (50 μg/ml) at 37°C for 4 h. The lipid raft/caveolae (lipid raft) and non-lipid raft microdomain (non-lipid raft) localization of TβR-I (a), TβR-II (b), and caveolin-1 (c) in treated cells were then determined by sucrose gradient ultracentrifugation, followed by Western blot analysis using antibodies to TβR-I, TβR-II, and caveolin-1. Treatment of cells with 7-DHC (50 μg/ml) diminished the amounts of TβR-I (a), TβR-II (b), and caveolin-1 (c) in fractions 4 and 5 (lipid raft), and fractions 7 and 8 (non-lipid raft), as marked by an open arrowhead. The fraction was compared to that in cells treated with cholesterol. The data are representative of a total of three independent analyses. The relative amounts of TβR-I and TβR-II in lipid rafts/caveolae (fractions 4 and 5) and non-lipid raft microdomains (fractions 7, 8, and 9) in cells treated with cholesterol or 7-DHC were quantified by densitometry. The total amount of TβR-I, TβR-II, or caveolin-1 in lipid rafts/cavelolae/non-lipid raft microdomains in cells treated with cholesterol was taken as 100%. The relative total amounts of TβR-I, TβR-II and caveolin-1 in lipid rafts/caveolae (lipid raft)/non-lipid raft microdomains (non-lipid raft) in cells treated with 7-DHC were estimated to be ~10%, and ~10% and ~15%, respectively. Cholesterol treatment did not appear to affect the total relative amounts of TβR-I, TβR-II and caveolin-1 in the microdomains.