Sections were washed twice with PBS, for 5 minutes at room temperature, and then washed once with PBS containing 0.2% Triton X-100 (PBS-Triton) for 5 minutes. Next, sections were incubated with blocking agent (5% goat serum diluted in PBS-Triton) for 1 hour at room temperature. Blocking agent was removed and sections were then incubated with Abcc3 primary antibody (diluted

1:100 in blocking agent) for 2 hours at room temperature. Sections were washed thrice with PBS-Triton and then incubated with Alexafluor 488 goat anti-rat IgG antibodies diluted 1:100 in PBS-Triton and Rhodamine-conjugated phalloidin (Invitrogen Inc., Carlsbad, CA; diluted 1:200) for 1 hour at room temperature in dark. After incubation, sections were washed twice with PBS-Triton, followed by a wash with PBS, and then double-deionized water. Sections were allowed to air dry and were

mounted JNJ-26481585 with Prolong® Gold containing DAPI (Invitrogen Inc., Carlsbad, CA). Acetaminophen (APAP) disposition in C57BKS and db/db male mice Ten week old C57BKS and db/db male mice (n = 5) were obtained from Jackson Laboratories (Bar Harbor, ME). Only male mice were used for this study, as both genders exhibited increased liver Abcc3 and 4 expressions, and APAP disposition studies in rodents are MRT67307 price typically performed using males. After two weeks acclimation, mice were administered APAP (100 mg/kg, po) in 0.9% saline. Immediately after dosing, mice were housed individually in metabolic cages equipped with

urine collection trays that kept cool with custom ice packs (Techniplast, USA). The total urine volume over 24 hrs was measured. To precipitate proteins buy LY2603618 in urine, samples (100 μl) were diluted with 200 μl cold Phenylethanolamine N-methyltransferase methanol and centrifugated at 4,000 g for 30 min at 4°C. The resulting supernatants were collected (250 μl) and diluted with 500 μl mobile phase. After mixed, the samples were centrifuged at 4,000 g for 10 min at 4°C. 100 μl of the supernatant is used for HPLC analysis. The column used for HPLC analysis was Eclipse XDB-C18 (4.6 mm x 15 cm, 3.5 μm). The mobile phase A contained 8% methanol and 1% acetic acid in water, and B contained 50% methanol in water. For first 5 min, mobile phase B was maintained at 100% followed by linear gradient of 10 min, ending in 25% of mobile phase B. Statistical analysis Statistically significant differences between groups were determined by one-way ANOVA followed by a Newman-Keuls post hoc test. Unless otherwise stated, all data is presented as mean ± SEM for n = eight mice per group. For APAP disposition data, t-test was used for statistical significance. Values with P≤0.05 were considered statistically significant. Acknowledgements We thank Dr. Michael Goedken, Dr. Maureen Drisoll and Dr. Jialin Xu for providing valuable inputs in editing the manuscript. We also thank Dr. Michael Goedken for pathological evaluation of H and E stained liver and kidney sections.