Mdck 2 cells

The generation of the ABCB4 mutants G68R, G228R, D459H and A934T has been previously described [13 (link),16 (link)]. Madin-Darby canine kidney MDCK-II cells and human embryonic kidney AD-293 cells were obtained from ATCC (LGC Standards Barcelona, Spain) and Agilent Technologies (Santa Clara, CA, USA), respectively. The mouse monoclonal anti-ABCB4 antibody (clone P3II-26) was purchased from Millipore (Billerica, Masachussets, USA). The rabbit anti-calnexin antibody was obtained from StressMarq (Victoria, Canada), and the anti-Na/K-ATPase antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-mouse AlexaFluor594-conjugated and anti-rabbit AlexaFluor488-conjugated secondary antibodies were from Molecular Probes (Eugene, OR, USA). Sodium 4-PBA, curcumin, cycloheximide, brefeldin A and standard lipids (phosphatidic acid, phosphatidylinositol, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin) were provided by Sigma (St. Louis, MO, USA). [³H]-choline (60–90 Ci/mmol) was purchased from Perkin Elmer (Massachusetts, USA). All other reagents were of analytical grade.

MDCK II cells (ATCC) were grown in alpha-MEM medium supplemented with 10% fetal bovine serum, 100 U ml⁻¹ pernicillin/streptomycin and 200 mM glutamine. Cells were transfected using Amaxa Kit L (Lonza) following the manufacturer's instructions.

Human lung adenocarcinoma epithelial A549 cells and Madin-Darby Canine Kidney (MDCK-II) cells (ATCC, Manassas, VA, USA) were grown in 6-well plates in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (PAA Laboratories GmbH, Goetzis, Austria Austria), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (P/S) (Gibco, Waltham, MA, USA) at 37 °C in a 5% CO₂ atmosphere and at 95% humidity. The pandemic influenza A/Hamburg/04/09 (H1N1pdm09) provided by the strain collection of the Institute of Medical Virology, Justus Liebig University, Giessen, Germany was propagated in MDCK-II cells in infection medium (DMEM containing 0.2% bovine albumin (BA) (PAA Laboratories GmbH, Cölbe, Germany), P/S, and 1 μg/mL TPCK-treated trypsin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)) at 37 °C, 5% CO₂, and 95% humidity. Binase was purified from Bacillus pumilus 7P culture fluid according to the procedure described in [70 (link)]. RNase activity of purified binase was 1.2 × 10⁶ units per milligram of protein.