Restriction and modifying enzymes

Fluorescein isothiocyanate-labeled inulin and canagliflozin (Invokana) were purchased from Sigma-Aldrich (Oakville, ON, Canada) and Janssen Inc. (Toronto, ON, Canada), respectively. Dulbecco Modified Eagle Medium (DMEM) (Cat. No. 11966-025), Ham’s F12 medium (Cat. No. 11765-054) and fetal bovine serum (FBS) were bought from Gibco (Thermo Fisher Scientific, Montreal, QC, Canada). Oligonucleotides were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA) and listed in Supplemental Table 1. Restriction and modifying enzymes were purchased from New England Biolabs (Whitby, ON, Canada). The sources of antibodies used are listed in Supplemental Table 2. HK-2 (an immortalized human renal proximal tubular cell line) (Cat. No. CRL-2190) was obtained from American Tissue Cell Collection (ATCC) (Manassas, VA) (http://www.atcc.org). Human SGLT2 gene promoter (N-1,986/+22) was amplified from HK-2 genomic DNA by PCR with specific primers (Supplemental Table 1) and then inserted into pGL4.20 reporter vector (Promega, Sunnyvale, CA) at Xho1and Bgl II restriction sites.

17β-Oestradiol (E2/oestrogen) and analytical grade reagents were obtained from Sigma-Aldrich (Dorset, U.K.); Δ⁴-dafachronic and (25S)-Δ⁷-dafachronic acids (DAs) were purchased from Cambridge Bioscience (Cambridge, U.K.) and Insight Biotechnology Ltd (Wembley, London, U.K.) respectively. Stock solutions of these ligands were prepared in sterile DMSO (Sigma-Aldrich) at 1000× concentrations (100 µM E2 or 1 mM DAs) and stored in aliquots at −20°C. Restriction and modifying enzymes were purchased from New England BioLabs (Hitchin, U.K.).