Fig. 4. Erlin-1 and erlin-2 are enriched in cellular membranes and lipid raft fractions. (A) U937, 3T3-pBabe, 3T3-erlin-1 and 3T3-erlin-2HA cells were subjected to hypotonic lysis. Membrane (m) and cytosolic (c) fractions were collected and equal proportions of each fraction (by fraction volume) were subjected to SDS-PAGE and western blotting for erlin-1 (with 10E6) and erlin-2HA (with 12CA5). Purity of the membrane and cytosolic fractions was assessed by western blotting for Src and pyruvate kinase (PK), respectively. (B) Lipid raft isolates were prepared from U937, 3T3-pBabe, 3T3-erlin-1, and 3T3-erlin-2HA cell lines as described. Soluble fractions were collected from the bottom of the sucrose gradients. Equal proportions of the lipid raft (r) and soluble (s) fractions were subjected to SDS-PAGE and western blotted for erlin-1 (using 10E6) and erlin-2HA (using 12CA5). Fraction purity of the raft fraction was assessed by western blotting for flotillin-1; purity of the soluble fraction was assessed by western blotting for CD71-transferrin receptor (TfnR).