The target profiles of the drugs are shown in Additional file 3. Figures 2 and

Briefly, after incubat ing the cells in the presence of MTT at 37 C for 1. 5 hours, the supernatant was discarded and the intracellular formazan was solubilized with 95% isopropanol 5% formic acid. Absorbance was read at 550 nm using a microplate reader. Cytotoxicity ABT-888 ic50 was determined in the kinetic experiments after 24 hr substrate exposure, thereby ensuring that a substrate exposure of up to 30 min will not encompass substrate cytotoxicity. Conversely, in the transport induc tion experiments cytotoxicity was determined after 24, 48, 72 and 96 hours of exposure. All assays were performed with the respective vehicle controls and cadmium chloride as positive control. Transporter induction experiments For transporter induction experiments, freshly isolated PKC cells were seeded on 6 well ThinCert PET tissue culture inserts in standard cul ture medium and medium was changed every 48 hours.

Cell cultures formed a tight monolayer on day 7 of cul ture, which was confirmed microscopically before use. At this time point medium was changed again Afatinib 分子量 and incu bations with potential transporter inducers were started by adding 50 nM or 1 uM of bromosulphophthalein, p aminohippuric acid, taurocholic acid, triiodothyronine, dehydroepiandrosteron sulfate, testosterone and dexame thasone to the basolateral compartment. After 48 hours of exposure, samples for RNA extraction were prepared by standard trypsin EDTA treatment followed by washing with PBS. Resulting cell pellets were snap frozen and stored at −80 C until use.

RNA extraction and RT PCR analysis Total RNA was prepared from cell pellets and minced pieces of tissue via the phenol chloroform method using AG-1478 価格 Trizol. Concentration and quality was esti mated via 260 280 nm values. Representative samples were tested for RNA integrity via denaturing electrophor esis. cDNA was prepared using 1 ug total RNA, Oligo 18 and M MuLV reverse transcriptase according to manufacturers instructions. PCR was performed using 2 ul cDNA sample, 2 × PCR Master Mix, specific primers. Amplification was performed with an initial denaturation step at 92 C for 3 min followed by 30 cycles of 94 C for 60 sec, 58 C for 60 sec and 72 C for 60 sec and a final extension step at 72 C for 7 min. PCR products were separated on 1. 5% agarose gels with 1 × TAE buffer and stained with ethidium bromide. A MultiDoc It digital imaging system was used for photographic documentation.

All nucleic acid analyses were performed with appropri ate control samples in parallel. PCR products were sent to MWG, MWG Biotech AG, Ebersberg, Germany for automated sequence analysis. Protein preparation and analysis Enriched membrane protein fractions were prepared according to Scalera et al. with modifications. Briefly, cell pellets or minced tissue pieces were resuspended in sucrose buffer, 1 mM EDTA, 1 mM PMSF using a ratio of ap proximately 1,5. Crude homogenates were prepared using a motor driven teflon potter followed by an ultra sound step at 4 C. Cellular debris was removed by centri fugation and the supernatants were centrifuged again. The pale frac tions of the pellets were combined with the supernatants and centrifuged.