Figure 6. Association of IR, cholesterol, SM and PC with DRMs.

(A) Isolated plasma membranes from WT and NPC mouse livers were solubilized in 1% Triton X-100 at 4 °C and subjected to Optiprep density-gradient centrifugation. Proteins from the gradient fractions were precipitated with trichloroacetic acid and analysed by Western blotting with anti-IRβ antibodies. OP, Optiprep. (B) For analysing the fraction of ligand-occupied IR in DRMs, WT and NPC hepatocytes were stimulated with ¹²⁵I-insulin, washed and subjected to density-gradient centrifugation. The amount of radioactivity in the fractions was determined and the percentage in DRMs is indicated. (C) The fraction of radiolabelled lipids in DRMs from WT and NPC hepatocytes. Black bars, [³H]PC; white bars, [¹⁴C]cholesterol; grey bars, [³H]SM. The cells were labelled and the lipids were analysed as detailed in the Experimental section. Values are means±S.D. for three to four independent experiments performed in triplicate; *P<0.05, **P<0.001.