Quantitation of intracellular LAMP2 and NPC1 protein

HeLa and HEK293T cells were grown to sub-confluence in DMEM supplemented with 7.5% FBS. One 10cm dish of cells was washed 3 times with cold PBS, then lysed with 500µl RIPA buffer with protease inhibitor cocktail (Sigma). After 30 min on ice, the lysate was centrifuged at 13,000 rpm for 15 min at 4°C. The resulting supernatant was transferred to a new tube and protein was measured by BCA assay. Lysates were resolved by SDS-PAGE, using different amounts of purified human LAMP2 or NPC1 protein as standards. After transfer to nitrocellulose, the blot was probed with anti-human LAMP2 or NPC1 antibody followed by IRDye 800CW labeled anti mouse (for LAMP2) or rabbit (for NPC1) secondary antibody, and visualized using a LI-COR Odyssey Imaging System and analyzed using ImageJ software. Calculations were based on molecular weights of 45,874 for LAMP2 and 142167 for NPC1 polypeptide chains, and neglected glycan contribution, which is not measured in the protein assay employed. Purified, full length NPC1 protein was the gift of Dr. Xiaochun Li (Rockefeller University) and was N-glycanase treated.