(A) Coomassie-stained SDS-PAGE of purified, secreted human LAMP1, LAMP2 or LAMP domains from LAMP2. B,C. 3H-cholesterol or 3H-25 hydroxycholesterol binding to soluble LAMP2 (full length protein). Also shown in C is binding in the presence of 50 µM cold hydroxycholesterol. D,E, 3H-cholesterol binding to indicated, soluble proteins compared with the soluble NPC1 N-terminal domain. (F), Cholesterol binding to LAMP2 domains 1 and 2 compared with full length, soluble LAMP2 protein. P values were determined relative to the full length soluble LAMP2 protein. (G) Sterol competition (30 µM) for 3H-cholesterol binding to soluble LAMP2. P values were determined relative to no cold addition. H, I. pH dependence and kinetics of 3H-cholesterol binding to LAMP2. In B,C,E,H and I, a representative experiment is shown; C and E show the average of duplicates. In C, the background counts in reactions containing the control protein, GFP-binding protein, were subtracted; in D and E, the control was TIP47 protein. D,F, and G show the combined results of two experiments in duplicate. Numbers at right (A) indicate mass in kD for this and all subsequent figures. Reactions contained D,E,G,H,I, 500 nM total cholesterol; F, 5 µM total cholesterol; B and C were carried out using increasing concentrations of the indicated sterol.