Fig. 2.

Role of the SSD within NPC1 in mediating the binding between NPC1 protein and [³H]AC. (A) Effect of NPC1 mutations within the sterol-sensing domain on [³H]AC labeling. CT43 cells (3.0 × 10⁶) or 25RA cells (6.0 × 10⁶) transiently transfected with WT or various mutant pNPC1-FP as indicated were photolabeled by using [³H]AC and processed for radioluminography (RL) and Western blotting (WB) according to procedures as described in Materials and Methods. The results using CT43 cells are representative of four experiments. The results using 25RA cells are representative of two experiments. (B) Percentage colocalization of NPC1-FP with LysoTracker. CT43 or 25RA cells were plated in wells with coverslips, grown in medium A, transiently transfected with various NPC1-FP constructs as indicated, and then grown in medium A for 48 h. Afterward, cells were stained with LysoTracker red and viewed under a confocal microscope according to procedures described in Materials and Methods. Red signal, LysoTracker; green signal, NPC1-FP. The yellow signal indicates colocalization of the red and green signals. Values given are the percentage colocalization between LysoTracker and a given NPC1-FP as indicated. To estimate percentage colocalization for each construct, results from six different images were averaged and quantified by using coollocalizer 1.1.2 (Cytolight). Only the results using CT43 cells were shown. The results expressing various NPC1 constructs in 25RA cells as the host were very similar to those reported in CT43 cells.