Lipofection and NPC1 Western blotting

COS cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA), and grown in Eagle medium with 10% fetal bovine serum. Cells were plated in 6-well dishes and lipofected with 2 μg/dish of either pCMV-NPC1 or pPDGFB-NPC1 and Lipofectamine 2000 (ThermoFisher, Carlsbad, CA). After 2 days of transfection, washed monolayers were lysed and separated by reducing SDS-PAGE followed by blotting to nitrocellulose with an iBlot2 (ThermoFisher). The filters were probed with a 1:1,000 dilution of a rabbit polyclonal antibody directed against the C-terminus of human NPC1 (NB400-148, Novus Biologicals, Littleton, CO), a sheep anti-rabbit secondary antibody from Bethyl Labs (Montgomery, TX), and the ABC Vectastain reagent (Vector Labs, Burlingame, CA), followed by color development with diaminobenzidine. High molecular weight standards were run in parallel.