Supplementary Materialspharmaceutics-11-00624-s001. excretion of S107 drug transporter substrates, potentially leading to drugCdisease interactions. = 23C24/group). This dose is usually well-tolerated by pregnant animals and a full immune response is usually elicited [17]. Rats were then anesthetized using isoflurane (Fresenius Kabi Canada, Toronto, ON, Canada) and euthanized at 6, 24, or 48 h after injection (= 7C8/group per time point). Kidneys were harvested, snap-frozen in liquid nitrogen and stored at ?80 C for further analysis. In order to attain statistically significant differences at the 95% confidence level based on variability and effect size seen in our pilot study, we calculated that a minimum of 6 animals/group were required per time point. All animal studies were conducted based on the guidelines of the Canadian Council on Animal Care and were pre-approved S107 by SARP2 the Office of Research Ethics at the University of Toronto, AUP #20011917, (Approved 20 February 2017, Last Renewed S107 12 March 2019). 2.2. RNA Extraction and Quantitative Real Time Polymerase Chain Reaction Total RNA was isolated from 50 mg of frozen renal tissue using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturers protocol. The purity and concentration of the RNA was decided using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Total extracted RNA (2 g) was treated with DNase (Invitrogen) and reversed transcribed to cDNA using a high capacity cDNA RT Kit (Applied Biosystems, Foster City, CA, USA). qRT-PCR was conducted using a Power SYBR Green detection system (ABI HT 7900; Applied Biosystems, Streetsville, ON, Canada) and samples were loaded in triplicates with primers specific for each gene (Supplementary Materials, Table S1). To be able to calculate the comparative mRNA degrees of each gene appealing, a comparative threshold routine technique (CT) was utilized. The expression of every gene was normalized towards the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Normalization with -actin led to similar outcomes. 2.3. Membrane Proteins Extraction and Traditional western Blot Evaluation Membrane proteins fractions had been extracted from tissue as previously referred to (23). Quickly, renal tissues (300 mg) was homogenized in lysis buffer (0.1 M Tris-HCL (Sigma Aldrich), pH 7.5), containing 3 L/mL protease inhibitor cocktail (Sigma Aldrich), and 50 S107 g/mL phenylmethylsulfonyl fluoride (Bioshop, Burlington, ON, Canada). Tissues lysate was centrifuged at 900 for 15 min at 4 C (Beckman Coulter, Mississauga, ON, Canada). The supernatant was centrifuged at 100,000 for 1 h at 4 C. Pellets had been resuspended in homogenizing buffer and proteins concentrations were assessed by Bradford assay (Bio-Rad Laboratories, Mississauga, ON, Canada). Total membrane protein (50 g) had been separated using 10% sodium dodecyl sulfate (SDS)-Web page and used in polyvinylidene fluoride membranes (Bio-Rad Laboratories). Membranes had been then obstructed with 5% dairy natural powder in tris-buffered saline with tween and incubated right away with the next major antibodies: anti-OCT2 (1:1000, Kitty# sc-365116), anti-MATE1 (1:200, Cat# sc-138983), anti-OAT3 (1:200, Cat# sc-293264), anti-ENT1 (1:100, Cat# sc-377283) (all purchased from Santa Cruz Biotechnology, Dallas, TX, USA), anti-OAT1 (1 g/mL, Cat# SAB2102177) (Sigma Aldrich), anti-URAT1 (1:1000, Cat# URAT11-A) (Alpha Diagnostic International, San Antonio, TX, USA), anti-P-gp (C-219; 1:100, Cat# ALX-801-002-C100) (Enzo Life Sciences, Farmingdale, NY, USA), and anti-PEPT2 (1:250, Cat# PA5-424800) (Thermo Fisher Scientific, Waltham, MA, USA). Membranes were then treated with secondary anti-mouse (1:30,000, Cat# NA931) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for P-gp, OCT2, MATE1, OAT3, ENT1, or secondary anti-rabbit (1:1000, Cat# NA934) (GE Healthcare, Mississauga, ON, Canada) for OAT1, OAT2, URAT1. Protein expression in each sample was normalized to the internal loading control -actin (1:75,000, Cat# A1978) (Sigma-Aldrich). A calibrator sample was also loaded on all gels to control for variability between gels. SuperSignal West Femto (ThermoScentific, Rockford, IL, USA) was applied to membranes for immunodetection. Band intensity was decided using Alpha Ease FC imaging software Version 6.0.0 (Alpha Innotech, Santa Clare, CA, USA). S107 2.4. Data and Statistical Analysis Data was analyzed using Prism software Version 6.0 (GraphPad Software Inc., La Jolla, CA, USA, www.graphpad.com). Students unpaired two-tailed 0.05) and returned to baseline by 24 h, as determined by ELISA [17]. Open in a separate window Physique 1 Poly I:C increases.