Effect of oxysterols on Cx43 phosphorylation and solubility in Triton X-100. LEC were incubated with 20 μg/ml cholesterol (lane 2), 7-keto (lane 3) or 25-OH (lane 4) for 3 hours. The Triton X-100 insoluble fractions of cell lysates were separated by SDS-PAGE, transferred to PVDF membranes and probed with antibodies directed against Cx43 (A). To allow quantification of phosphorylated forms of Cx43 the film of was overexposed (B). Actin on the same samples is included to demonstrate comparable loading of the lanes (C). The intensity of the bands was determined by laser scanning of the films followed by quantitative densitometric analysis and the results obtained are depicted in a graph (D and E). The values are the average of three individual experiments ± SD. * – indicates statistically significant differences from controls (p < 0.05).