Immunofluorescence microscopy and FACS analysis of NTCP binding with pre-S1 peptides Plasmid encoding human, or Tupaia NTCP, with or without a tag, or a control plasmid was transfected into cells. The cells were washed and blocked with 3% BSA 24-36 hr after transfection and then stained with biotin-labeled peptides at 4°C about 1-2 hr, followed by fixation with 4% paraformaldehyde (PFA) for 10 min. Cells were then stained with PE-labeled Streptavidin (eBioscience). In some cases, Myr-59 peptide containing the first 59 residues of pre-S1 domain with an N-terminal myristoylation modification and labeled with FITC (FITC-pre-S1) was applied to cells directly. The cell images were captured with a Nikon Eclipse Ti Fluorescence Microscope or a Zeiss LSM 510 Meta Confocal Microscope. For FACS analysis, the FITC-pre-S1 stained cells without fixation were detached with 0.5 mM EDTA/PBS, washed and resuspended in PBS, and analyzed with a FACS LSRII instrument (BD).