Figure 5.

Subcellular localisation of scavenger receptor class B type I (SR-BI) in enterocytes. (A) Small intestinal mucosa was fractionated into Mg²⁺ precipitated membranes (Mg), microvillar membranes (Mic), and soluble protein (Sol), as described in the methods section. Samples of the three subcellular fractions proportional to their relative amount in the homogenate (about 50–200 μg of protein) were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis. After electrophoresis and transfer onto Immobilon, the 82 kDa band of SR-BI was visualised by immunoblotting. After blotting, total protein was stained by Coomassie brilliant blue. (B) Immunopurification of SR-BI. Microvillar membranes (1) were solubilised by 1% Triton X-100 at 20°C (both raft and non-raft membranes are solubilised at this temperature), and preincubated with protein A-Sepharose, followed either by anti-SR-BI antibodies coupled to protein A-Sepharose (2) or free protein A-Sepharose (3), as described in the methods section. Samples were analysed by immunoblotting as described above. (The broad band of 50 kDa in lane 2 represents the SR-BI antibody. In lane 3, protein A-Sepharose bound some endogeneous immunoglobulin and secretory component.)