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4 mM benzamidine) and incubated for 15 min at 4��C. The suspensions, adjusted to 40% (w/v) sucrose by adding 2 ml of ice-cold 80% sucrose (w/v) in MBS, were placed at the bottom of an ultracentrifuge tube: two equal volume oxyclozanide layers of 30 and 5% sucrose were added on top, and the gradients were centrifuged at 100,000 �� g for 20 h. The light opalescent band confined to the 5�C30% interface was collected, diluted three times with MBS and centrifuged at 100,000 �� g for 1 h. The fractions thus obtained were resuspended in buffer B, frozen in liquid nitrogen and stored at ?80��C until use. SDS/PAGE and Western blot analysis Membrane preparations were heat-denatured in SDS-containing sample buffer with reducing agents and loaded onto Tris-glycine 4�C12% acrylamide gradient SDS-containing gels. Following electrophoresis at 100 V, proteins were transferred to nitrocellulose membranes at 100 V for 1 h. Membranes, blocked with 5% non-fat dry milk in Tris-saline buffer (mM: 140 NaCl, 20 Tris-HCl, pH 7.6) plus 0.2% Tween 20 were probed with specific antibodies directed against T-tubule or SR proteins. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit hypoxia-inducible factor pathway IgG were used as secondary antibodies. Immunoblots were developed using an ECL kit and automated image capture was performed with the ChemiDoc system (Bio-Rad, Hercules, CA). All blot quantifications involved measuring band intensities; the amount of protein loaded in each lane was checked by Coomassie blue staining. Other procedures NOX activity was determined as described previously (Sanchez et al., 2008). Protein concentration was determined by the BCA protein assay using commercial BSA as a standard. Cholesterol content was determined using the colorimetric total cholesterol kit or the fluorescence Amplex Red Cholesterol Assay kit, as detailed in the text. Electron microscopy (EM) analysis of DRM fractions was performed as described (Badizadegan et al., 2000). Statistical analysis To detect significant differences between two groups, statistical analysis http://www.selleckchem.com/products/sorafenib.html of data, presented as Mean �� SE, was determined with the Student's t-test (two-tailed); a value of p