Hypermethylation of the CaSR and VDR genes in the parathyroid glands in chronic kidney disease rats with high phosphate diet

Journal name: - Human Cell

Taketo Uchiyama1, 2, Norifumi Tatsumi1, Sahoko Kamejima1, 2, Tsuyoshi Waku3, Ichiro Ohkido2, Keitaro Yokoyama2, Takashi Yokoo2, Masataka Okabe1,*.

1Department of Anatomy, The Jikei University School of Medicine, Tokyo, Japan

2Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan

3Laboratory for Genetic Code, Graduate School of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan

*Correspondence: Masataka Okabe. Tel: +81 3 3433 1111. Fax: +81 3 3433 2065. Email address:

Supplemental Materials and Methods

Western blotting

Protein was isolated from rat PTG (positive control) and human embryonic kidney (HEK 293) cells (negative control) [S1, S2], which were homogenized in 0.1 ml ofRIPA buffer. Protein concentrations were determined using the NanoDrop spectrophotpmeter (Thermo Fisher Scientific, Japan). All samples (containing 15µg protein) were mixed with loading buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins in the samples from rat PTG and HEK 293 cells were separated and transferred onto polyvinylidene difluoride membranes by electroblotting (200 mA for 30 min). The membranes were blocked in phosphate-buffered saline with 0.1% Tween 20 containing 0.5% skimmed milk at room temperature for 1 h. Membranes were then incubated overnight at 4C with antibodies against CaSR (1:200), VDR (1:200), β-actin (SC-47778; 1:1000; Santa Cruz Biotechnology Inc., CA, USA). Membranes were then washed with PBST three times for 15min and incubated with an alkaline phosphatese-conjugated goat anti-mouse and anti-rabbit secondary antibody (1:1000) in PBST for 1 h at room temperature. The densities of the protein bands were quantified using a ChemiDoc XRS imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with β-actin serving as an internal control.

References

[S1] Matsunawa M, Akagi D, Makishima M, Vitamin D receptor activation enhances Benzo[a]pyrene metabolism via CYP1A1 expression in macrophages, Drug Metab Dispos. 40 (2012) 2059-2066.

[S2] Bai M, Trivedi S, Brown EM, Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293 cells, J Biol. Chem. 273 (1998) 23605-23610.