Alexa 594 conjugated transferrin

The following antibodies were used for immunofluorescence staining or western blot analyses: anti-FoxA2 (used at 1:400, Cell Signaling), anti-HNF4α (used at 1:500, Cell Signaling), anti-AFP (used at 1:1000, Sigma-Aldrich), anti-ALB (used at 1:1000, Cedarlane, Burlington, Canada), anti-ZO-1 (used at 1:1000, Thermo Fisher), anti-E cadherin (used at 1:500, Cell Signaling), anti-SR-BI (used at 1:100, Novus Biologicals), anti-BCRP (used at 1:100, Millipore), anti-MRP2 EAG5^{48 (link)} (used at 1:200, a kind gift from Anne Nies, IKP Stuttgart^{48 (link)}), anti-Apo-CIII (used at 1:500, Abcam), anti-CYP8B1 (used at 1:100, Abcam), anti-ORF2 (used at 1:400, a kind gift from Suzanne U. Emerson, NIH) and anti-HAV capsid (used at 1:1000, a kind gift from Stanley M. Lemon, UNC School of Medicine). Alexa Fluor 488 and 549 anti-mouse (used at 1:1000) and Alexa Fluor 488 and 549 anti-rabbit (used at 1:1000) antibodies were purchased from ThermoFisher. Alexa 594-conjugated transferrin was purchased from ThermoFisher. Tenofovir, Tenofovir disoproxil fumarate, and Emtricitabine were obtained through the AIDS Reagent Program, Division of AIDS, NIAID, NIH. Elvitegravir and Cobicistat were purchased from SelleckChem. Sofosbuvir was purchased from Acme Bioscience. BX795, oleic acid and lomitapide were purchased from Sigma-Aldrich.

Huh-7.5 cells seeded on 8-well chamber slides were treated with DMEM supplemented with the indicated inhibitors for 1 hr. Cells were then rinsed, placed on ice for 10 min, and incubated with 10–25 µg.ml⁻¹ Alexa 594-conjugated Transferrin (Thermo Fisher, #T13343) or cholera toxin subunit B (Thermo Fisher, #34777) diluted in supplemented DMEM for 15–20 min at 37˚C. Cells were then fixed with 4% paraformaldehyde (PFA) and nuclei were counterstained with 300 nM DAPI.