Ab108921

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.

Cells were homogenized in lysis buffer (50 mM Tris‐HCl pH = 8.0, 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P‐40 supplemented with proteinase inhibitors), resuspended and centrifuged at 1000 g for 10 min at 4°C. The supernatants’ protein contents were quantified, separated on 4%–12% precast gels Bis‐Tris (NuPAGE, Invitrogen, Carlsbad, CA) subjected to SDS‐PAGE (20 µg of total protein) and semi‐dry transferred to nitrocellulose membranes (Whatman, GE Healthcare, Marlborough, MA). For enzymatic deglycosylation of proteins, cell extracts were incubated in the presence or absence of 1 unit of PNGase F and Endo H (NEB, Ipswich, MA) for 1 h at 37°C according to the manufacturer's instructions. NPC1 protein was detected by incubation with anti‐NPC1 rabbit polyclonal antibody (ab108921, Abcam, Burlingame, CA) overnight and horseradish peroxidase (HRP)—conjugated IgG as the secondary antibody (sc 2313, Santa Cruz Technologies, Santa Cruz, CA). Signal was developed using enhanced chemiluminescence (ECL) (GE Healthcare, Marlborough, MA) and visualized on a ChemiDoc™ XRS⁺ (Bio‐Rad, Hercules, CA). Band intensity was calculated in Image Lab software version 6.0 (Bio‐Rad, Hercules, CA), after background subtraction and are expressed as Endo H‐sensitive/Endo H‐resistant ratios.