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Fig. 2. Distribution of SIRP{alpha} in lipid microdomains. Growth cone particles (GCPs) were treated with different detergents (indicated on the left of each panel) and fractionated in sucrose density gradients (A-E); the sucrose concentrations of the fractions are given at the top of each lane. Fractions were collected from top to bottom of the gradients. Fractions were analyzed by western blot with the indicated antibodies (IB, right; {alpha}=anti). (C) The experiment without detergent serves as a control for non-solubilizing conditions. Anti-transferrin receptor (TfR) blot is a control for Brij 98, showing that transmembrane proteins such as TfR are solubilized under these conditions. (D) Saponin was added before the Brij 98 extraction of GCPs to disrupt cholesterol interactions. The controls without saponin treatment produced the same results as in C and, therefore, are not shown again. (E) MßCD was used to extract cholesterol. In these experiments, samples of different density (range indicated on the top) were pooled to represent high-, middle- and low-density fractions. For detailed description, see Results.