Fig. 4. Fractionation of MDCK cells in iodixanol gradients. MDCK cells were homogenized either 6 hours or 48 hours after induction of calcium-dependent cell-cell adhesion. Post-nuclear supernatants were mixed with 10%, 20% and 30% (w/v) iodixanol, layered step-wise in centrifuge tubes and centrifuged at 350,000 g for 3 hours. The presence of Sec8, ZO-1, ZO-2, afadin, occludin, claudin-1, claudin-2, E-cadherin, nectin-1 and nectin-2 in gradient fractions was assayed by SDS-PAGE followed by immunoblotting with specific antibodies. Protein levels were quantified using a Molecular Dynamics Phosphorimager. Densities of each fraction were calculated after measuring refractive indices with a refractometer, and are plotted as dotted lines on each graph with values (in g/ml) indicated on the y-axis.