Figure 4.

Gradient density floatation of the hSlo-N200A in transfected MDCK cells. To assess raft association of hSlo-N200A, transfected MDCK cells were extracted either with 1% Triton X-100 at 4°C (A) or 500 mM sodium carbonate pH (B). Lysates were centrifuged to equilibrium in a linear sucrose gradient to isolate lipid rafts by floatation. Fractions were harvested from the bottom of the gradient, and aliquots were precipitated with 20% trichloroacetic acid and subjected to SDS/PAGE and immunoblot analysis. Proteins were visualized by ECL. (A) hSlo-N200A was detected floating in lighter buoyant densities together with the endogenous raft-marker caveolin-1. (B) High-salt treatment did not prevent floatation of hSlo-N200A that occurred mostly in fractions 3 and 4. The bulk of caveolin-1 is seen floating in fractions 5 and 6. Na⁺/K⁺ ATPase, used as a negative control of no association with lipid rafts, did not float and was recovered at the bottom of the gradient.