U18666A alters the detergent solubility of GPI-anchored proteins in late endosomes.

<p>A: DRMs were prepared from late endosomes (BHK cells +/− U18666A) by flotation on an OptiPrep gradient. Six fractions were collected from the top and analyzed by immunoblotting for the presence of flotillin-1 (Flot-1), Rab7, Lamp-1, annexin II (AnnII) and MLN64. B: BHK cells treated or not with U18666A. Total cells or purified late endosomes were solubilized in 1% Triton ×100 at 4°C (as in A and all other figures) or 37°C. The lysats were submitted to flotation on Optiprep gradients, 6 fractions collected and analyzed for the distribution of GPI-anchored proteins by aerolysin overlay. C: For the 4 conditions illustrated in B and for 3 independent experiments, the total GPI content of each fraction was quantified by densitometry and normalized to the total GPI-content of the gradient. Error bars represent the standard deviation (n = 3). * represent statistically significant differences (p<0.05).