Figure 2

Effect of oxysterols on Cx43 stability at the plasma membrane. The total amount of Cx43 following treatment with 20 μg/ml cholesterol, 7-keto or 25-OH, for 3 hours, was determined by western blot using antibodies directed against Cx43 (A). Actin on the same samples is included to demonstrate comparable loading of the lanes (B). LEC were treated with 20 μg/ml cholesterol (lane 3), 7-keto (lane 5) or 25-OH (lane 7) for 30 min prior biotinylation. Cells incubated in 0.2% ethanol were used as controls (lane 1). To determine the stability of Cx43 at the plasma membrane, the biotinylated cells were returned to the incubator for an additional 2 hours in DMEM, in the absence (lane 2) or presence of cholesterol (lane 4), 7-keto (lane 6) or 25-OH (lane 8). After extraction in situ by incubation in DPBS containing 0.5% Triton X-100, the biotinylated surface Cx43 was detected with antibodies directed against Cx43 following isolation with Neutravidin beads (C). The results obtained are depicted in a graph and correspond to the Cx43 remaining at the plasma membrane 2 hours after biotinylation (D). Each bar represents means ± SD of three independent experiments. * – indicates statistically significant differences from controls (p < 0.01).