Anti transferrin receptor

Mouse anti-caveolin-1 (#610406), anti-transferrin receptor (# 612124), anti-flotillin-1 (#610820), anti-Fas-ligand (#556372 clone NOK-1), anti-Fas-ligand (#556387 clone G247-4), anti-fyn (#610163), and rabbit anti-caveolin-1 (#610059) antibodies were purchased from BD Biosciences (San Diego, CA, USA). Cholera toxin, peroxidase conjugate (#227041), and mouse anti-GST antibodies (#OB03 clone 8-326) were from Calbiochem. Mouse anti-Fas antibodies (#610198) were from BD Transduction Laboratories, USA. Rabbit anti-beta-tubulin antibodies (#2146) were from Cell Signaling Technology, mouse anti-caspase 8 antibodies (#551242) were from BD Pharmingen. Purified goat anti-mouse (#170-6516) and anti-rabbit (#170-6515) IgG (H+L) horseradish peroxidase conjugates were obtained from Bio-Rad (Hercules, CA, USA). Alexa Fluor® 610-R-phycoerythrin goat anti-mouse (#A20980) and fluorescein goat anti-rabbit (#F2765) IgG (H+L) were from Invitrogen (Oregon, USA).
The following cell cultures were used: human cervical adenocarcinoma HeLa cells and african green monkey kidney cells COS-1. Сell lines were from Specialist collection of continuous cell lines of vertebrates (Institute of Cytology, Russian Academy of Sciences, St-Petersburg, Russia). Cells were maintained at 37 °C and 5% CO₂ in DMEM (Gibco) with 10% FCS (Hyclone).

Cells were detached with 5 mM EDTA in PBS and fixed with 4% formaldehyde before incubation with either anti-NTCP (9A8) or anti-transferrin receptor (Genetex) antibodies at 4° C. Cells were then stained with PE-conjugated secondary antibody and analyzed using a FACSCanto II instrument (BD Biosciences). For the drug screen, cells were treated with candidate compounds (50 μM) 24 hours before analysis. Data were analyzed with FlowJo software (TreeStar).