Fig 4. Cross-linking of 1519 to LAMP1 D1 is inhibited by cholesterol and vice versa.

(A) A photoreactive cholesterol analog (photoclick cholesterol) cross-linking to purified LAMP1 D1 is competed by a molar excess of cholesterol or 3.3 but not by a molar excess of epicholesterol or 102. Purified LAMP1 D1-His was incubated with the indicated compounds for 1h at 37°C prior to UV irradiation and click chemistry with an AF 488 azide. Labeled protein was detected by immunoblot with an anti-AF 488 antibody. Input LAMP1 D1 was detected using an anti-His antibody. (B) 1519 cross-links to purified LAMP1 D1 in a manner which is competed by a molar excess of 3.3 but not the inactive derivative, 102. Purified LAMP1 D1-His was incubated with the indicated compounds for 1h at 37°C prior to UV irradiation and click chemistry with an AF 488 azide. Labeled protein was detected by immunoblot with an anti-AF 488 antibody. Input LAMP1 D1 was detected using an anti-His antibody. (C) 1519 cross-links to purified LAMP1 D1 in a manner which is competed by a molar excess of 3.3 and cholesterol but not epicholesterol. Purified LAMP1 D1-His was incubated with the indicated concentrations of 1519 and 3.3, cholesterol or epicholesterol for 1h at 37°C prior to UV irradiation and click chemistry with an AF 488 azide. Labeled protein was detected by immunoblot with an anti-AF 488 antibody and input LAMP1 D1 was detected using an anti-His antibody.