Ab182099

The primary antibodies used for Western blotting or immunohistochemistry were monoclonal β-actin (AC-15, 1:1000; Santa Cruz), FGF23 (bs-5768R, 1:200; Bioss), αKlotho (NBP1-76511, 1:500; Novus Biologicals), NHERF1 (A-7, 1:200; Santa Cruz), NaPi-2a (ab182099, 1:500; Abcam), and NaPi-2a (ab151129, 1:500; Abcam), as described previously (Kang et al., 2017 (link)). Antigen retrieval for IHC was performed in Tris/EDTA (pH 9.0) for 2–3 min. IHC was performed with the following antibodies at 25°C for 2 hr: α-SMA (ab5694, 1:100; Abcam), NaPi-2b (S ab182099, 1:100; Abcam), TMPV6 (NBP2-32372, 1:100; Novus Biologicals), MINPP1 (LS-C368910, 1: 100; LSBio), or FGF23 (bs-5768R, 1:200; Bioss), Horseradish peroxidase-conjugated secondary antibodies were detected with 3,3,-diamino-benzidine tetrahydrochloride substrate (Dako, Glostrup, Denmark). All sections were counterstained with hematoxylin (Themo Scientific).

Harvested kidneys were homogenized in RIPA buffer (1% Tween 20, 0.1% SDS, 150 nM NaCl, 10 mM Tris-HCl (pH 7.4), 0.25 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM Na3VO4, 5 mM NaF (Sigma)). Lysates were collected by centrifugation at 15,000 rpm at 4 °C for 10 min. Equivalent amounts of protein were separated by SDS/PAGE and transferred to a PVDF membrane (EMD Millipore Corporation). Proteins were detected using anti-Klotho (ab98111, abcam, Cambridge, UK), anti-NaPi-IIa (ab182099, abcam) and anti-Actin (A2066, Sigma) antibodies. Bands were visualized using ECL Western Blotting Detection Reagent (GE Healthcare, Uppsala, Sweden).