For cell surface expression, the transfected cells expressing NTCPs or mutants were surfacebiotinylated with sulfo-NHS-LC-biotin (Pierce) following the manufacturer's instruction manual. The biotinylated cells were then lysed in 600 μl of 1× RIPA buffer supplemented with 1× protease inhibitor cocktail (Roche) and then treated with PNGaes F. Total cellular protein in the lysate was determined by using Bio-Rad DC Protein assay. About 300 μg Streptavidin T1 Dynabeads (Invitrogen) were then used to pull down surface-biotinylated proteins in the supernatants containing ∼160 μg of total cellular protein each sample. After extensive washing with 1× RIPA buffer, bound proteins were eluted, and separated by SDS-PAGE and subsequently examined with anti-C9 mAb 1D4 by Western blotting. For total NTCP expression, the same transfected cells expressing wild-type or mutant NTCPs were lysed with 1× RIPA buffer and treated with PNGase F. Each sample containing same amount of total cellular protein (∼8 μg) were loaded for SDS-PAGE followed by Western blotting analysis with mAb 1D4 that recognizes the C9 tag fused at the C-terminus of NTCPs.