Fig. 5.

Photolabelings of NPC1-YFP in vitro and protections against labeling by various agents. (A) The membrane fractions from CT60-NPC1-YFP cells were preincubated in buffer, in the presence [center and right lanes in the Western blotting (WB; Left) panel; filled and open circles in the gel slicing (Right) panel] or absence (left lane in the WB panel; open triangles in the gel slicing panel) of 50 mM CD for 20 min at 30°C, then photolabeled with 5 mM CD complexed with [³H]AC, according to procedures described in Materials and Methods. The labeled samples were subjected to IP with mouse monoclonal anti-GFP antibody (left and center lanes in the WB panel; open triangles and filled circles in the gel slicing panel), or with control mouse IgGs (left lane in the WB panel; open circles in the gel slicing panel). Western blotting with anti-GFP antibodies or gel slicing was performed according to procedures described in Materials and Methods. Results shown are representative of two experiments. (B) The membrane fractions from CT60-NPC1-YFP cells were preincubated for 20 min at 30°C in buffer containing 50 mM CD only (lane 1; CD only), or 50 mM CD complexed with an unlabeled steroid as indicated (lanes 2–4). Lanes: 2, cholesterol (5α-cholesten-3β-ol); 3, epicholesterol (5α-cholesten-3α-ol); 4, 5α-cholestan. Afterward, 5 mM CD complexed with [³H]-AC was added, and the samples were photolabeled, according to procedures described in Materials and Methods. Raw radioactivity was the sum of the radioactivity of gel slices 3–5. Results from WB were quantified by using nih image 1.63; the values obtained are shown at the bottom of each lane. The assays were done in duplicate; the results of the duplicates are shown. (C) Normalized radioactivity. The values were obtained by dividing the raw radioactivities by the mean pixel intensities shown in B. The results shown were the averages of duplicate assays. Results shown in B and C are representative of two experiments.