Polyscreen membrane

Electrophoresis was performed on 4–12% NuPAGE Bis-Tris polyacrylamide gels (Invitrogen, France). After size separation within the gel, proteins were transferred to a polyvinylidenedifluoride (PVDF) membrane (Polyscreen® membrane, Perkin Elmer, France). Membranes were blocked with a solution containing 0.1% Tween 20 and 200 mM Tris buffered solution (TTBS) complemented with 5% non-fat dry milk during 30 min and incubated with mouse β amyloid 1–16 (6E10; Eurogentec, France) monoclonal antibody at 4 °C overnight under gentle agitation. Incubation with the secondary fluorescent-conjugated antibody was performed during 1 h at room temperature. After 3 washes with TTBS and one with PBS, the membrane was scanned using a Licor Aerius automated infrared imaging system according to manufacturer’s instructions.

A total of 15 µg of total proteins extracted from the ileal mucosa, the liver, or the kidney were prepared in ice-cold buffer (0.154 M KCl, 0.01 M phosphate buffer, pH = 7.4), separated on 10% SDS-PAGE, then blotted onto a Polyscreen membrane (PerkinElmer Life Sciences). Rabbit polyclonal anti-ASBT, NTCP, BSEP, MRP2, OSTα, and OSTβ were a generous gift from Dr. M. Souidi (Institut de Radioprotection, Fontenay-aux-Roses, France), Prof. M. Trauner (Gastroenterology and Hepatology, Vienna, Austria), Prof. N. Ballatori University of Rochester School of Medicine, Rochester, NY, USA), and Prof. G. Kullak-Ublick (University Hospital Zurich, Zurich, Switzerland). Anti-b-actin antibodies and anti-rabbit peroxidase-conjugated secondary antibodies were purchased from Sigma-Aldrich. ECL blotting kit (PerkinElmer Life Sciences) was used for detection. Blot quantification was performed using calibrated GS-800 densitometer (Biorad, Marnes-la-coquette, France).