Calu-3 and NHBE cell

layers were harvested from Transwell® inserts on the same day as 3H-digoxin permeability experiments. mRNA isolation and cDNA synthesis were performed as described previously [26]. Manual TaqMan® analysis of the ABCC7 and ABCC10-12 genes was performed in triplicate in a 25 μl reaction mixture containing 30 ng cDNA, TaqMan® Universal PCR Master Mix (containing AmpliTaq Gold DNA polymerase, dNTPs with dUTP, passive reference and optimised buffer) and Assay-on-demand™ gene expression assay mix (containing 18 μM random hexamer primers). All other genes investigated were analysed via automated Taqman® PCR low density arrays using custom designed 384-well cards as described previously [26]. Amplification curves Buparlisib were analysed using the SDS2.1 software (Applied Biosystems, Foster City, CA) and thresholds for generation of selleck chemical C  T data were calculated automatically by the software. Target genes were compared with the two house-keeping genes RPLP0 (Large Ribosomal Protein) and MVP (Major Vault Protein) ΔC  T and assigned arbitrary categories for relative gene expression levels based on the 2T-ΔC value, i.e. relative expression levels >0.5 were considered as ‘high’ (+++), 0.02–0.5 as ‘moderate’ (++), 0.001–0.02 as ‘low’ (+) and <0.001

as ‘negligible’ (−). Cells were detached from the surface of the filters/flasks by the addition of 500 μl non-enzymatic cell dissociation buffer prepared in HBSS without calcium and magnesium salts. Cells were counted and resuspended in RIPA cell lysis buffer containing 1 μl of protease inhibitor cocktail set II per 200 μl (ratio of 20 million cells per 1 ml buffer solution) and agitated at 700 rpm at 4 °C for 30 min. Cell debris was pelleted at room temperature by centrifugation at 12,000g for 20 min and the inhibitors resulting supernatant decanted. Protein concentration was quantified using the RC DC™ protein assay (BioRad, Hemel Hempstead,

Hertfordshire). Protein samples were resolved using 7% Tris–acrylamide gels. Briefly, 10 μl of cell lysate solution containing 20–30 μg Methisazone of protein was diluted 1:1 with reducing sample buffer. Samples were run under denatured and reduced conditions alongside 5 μl precision plus protein standards (BioRad, Hemel Hempstead, UK) and resolved at 0.04 amps in running buffer. Transfer to a nitrocellulose membrane was conducted for 60 min at 100 V and at a temperature of 4 °C. Proteins transferred onto Western blots were visualised by staining with copper phthalocyanine 3,4′,4″,4″′-tetrasulphonic acid tetrasodium salt (CPTA). Samples were probed for the presence of MDR1 protein using 5 μg/ml of the mouse anti P-glycoprotein C219 primary antibody (Calbiochem, Nottingham, UK) for 16 h at 4 °C. All steps were performed using a chemiluminescence detection kit according to the manufacturer’s instructions (Invitrogen, Paisley, UK).