Cholesterol Trafficking and Lysosomal Analysis

Chemical and standard reagents including filipin and NBD-cholesterol were obtained from Sigma-Aldrich, St. Louis, MO. LysoTracker and fluorescent dextran were obtained from Life Technologies (Grand Island, NY). shRNA constructs were obtained from SA Biosciences (Valencia, CA). Antibodies against tubulin [1:100 for immunofluorescence (IF), T6199, Sigma-Aldrich], EGFP (1:100 for IF, 632380, Clontech) and mCherry (1:100 for IF, NBP1-96752, Novus Biologicals) have been described previously (Whyte et al., 2008 (link); Towns et al., 2009 (link)). Antibodies against StARD9 [1:100 for western blotting (WB), HPA014562] were obtained from Sigma-Aldrich. Site-directed mutagenesis utilized the Phusion kit (New England Biolabs, Ipswich, MA). Antibodies against NPC1 (5 μg/ml for IF, 1 μg/ml for WB, ab108921), LAMP1 (1:10 for IF, ab25630), SREBP1 (1:100 for IF, ab28481) and cathepsin B (1 μg/ml for IF, ab6313) were obtained from Abcam (Boston, MA). filipin (f9765) was obtained from Sigma-Aldrich. NBD-cholesterol (N1148) was obtained from Invitrogen (Waltham, MA). BacMAM-ER (C10590) was obtained from Thermo Fisher Scientific (Waltham, MA). Nocodazole (M1404) and taxol (T7402) were obtained from Sigma-Aldrich. The sequences of oligonucleotides used in this study are provided in Table S3.