Cholesterol Depletion for Membrane Diffusion

At 24 h posttransfection, cells were subjected to metabolic cholesterol depletion by incubation (18 h) with 50 μM compactin and 50 μM mevalonate (both from Sigma-Aldrich) in medium supplemented with 10% lipoprotein-deficient fetal calf serum following established procedures (Lin et al., 1998 (link); Shvartsman et al., 2006 (link)). This treatment reduces cholesterol by 30–33% (Eisenberg et al., 2006 (link); Shvartsman et al., 2006 (link); present results), leading to a selective increase in the lateral diffusion of raft-associated proteins without affecting the general biophysical properties of the plasma membrane. Nystatin treatment (25 μg/ml) was conducted in HBSS/HEPES/BSA and initiated 60 min (37°C) before treatment with TGF-β1 (Schnitzer et al., 1994 (link); Di Guglielmo et al., 2003 (link); Mitchell et al., 2004 (link)).