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Volume 62, Issue 3, Pages 757-762 (September 2002) Decreased sulfotransferase SULT1C2 gene expression in DPT-induced polycystic kidney Kazunobu Sugimura, Tomoaki Tanaka, Yoshihiko Tanaka, Haruna Takano, Kenji Kanagawa, Nobuyoshi Sakamoto, Shin-Ichi Ikemoto, Hidenori Kawashima, Tatsuya Nakatani Kidney International Volume 62, Issue 3, Pages 757-762 (September 2002) DOI: 10.1046/j.1523-1755.2002.00512.x Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 1 Representative photomicrograph (hematoxylin and eosin stain, corticomedullary junction) of a polycystic kidney induced by feeding of 1% 2-amino-4,5-diphenylthiazole (DPT) to Sprague-Dawley (SD) rats for two weeks. Kidney International 2002 62, 757-762DOI: (10.1046/j.1523-1755.2002.00512.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 2 Nucleotide sequence of the mouse SULT1C2 cDNA and the predicted amino acid sequence. The accession number of this cDNA in the GenBank is AY005469. Kidney International 2002 62, 757-762DOI: (10.1046/j.1523-1755.2002.00512.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 3 Northern blot analysis of mouse SULT1C2 mRNA in various adult tissues. Northern blot with poly(A)+ RNA from brain (lane 1), heart (2), kidney (3), liver (4), lung (5), muscle (6), skin (7), small intestine (8), spleen (9), stomach (10), testis (11) and thymus (12) was hybridized with 32P-labeled mouse SULT1C2 cDNA probe. The same membrane was hybridized with mouse G3PDH cDNA. Each lane contained 2 μg of poly(A)+ RNA. Kidney International 2002 62, 757-762DOI: (10.1046/j.1523-1755.2002.00512.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 4 Northern blot analysis of SULT1C2 mRNA in rat kidneys after DPT feeding. Kidneys were obtained from SD rats fed with 1% DPT for 1, 2, 4 and 14 days. Each lane was loaded with sample from one rat, and two sets of days 0 to 4 shown are from a different series of the experiments. The same membranes were hybridized with mouse G3PDH cDNA. Each lane contained 20 μg of total RNA. Kidney International 2002 62, 757-762DOI: (10.1046/j.1523-1755.2002.00512.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 5 Quantitative analysis of SULT1C2 mRNA in rat kidney and stomach after DPT feeding for one week. Northern blot analysis was done as described in the Methods section, and the signals quantified by a Bioimaging Analyzer were normalized with G3PDH. The mean ratio of control rats was taken as 100%. Kidney International 2002 62, 757-762DOI: (10.1046/j.1523-1755.2002.00512.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 6 SDS-PAGE of recombinant SULT1C2-His. SULT1C2-His was expressed as described in the Methods section, and purified by Ni2+-NTA agarose chromatography. Lane 1; flow through from Ni2+-NTA agarose column. Lane 2; purified SULT1C2-His. Kidney International 2002 62, 757-762DOI: (10.1046/j.1523-1755.2002.00512.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 7 Kinetic analysis of sulfotransferase activity top-nitrophenol by purified recombinant SULT1C2-His. (A) Plot of reaction velocity versus p-nitrophenol concentration as a substrate. (B) A Lineweaver-Burk double reciprocal plot of the data was used to determine the Km (3.1 mmol/L) and Vmax (20 nmol/min · mg). Kidney International 2002 62, 757-762DOI: (10.1046/j.1523-1755.2002.00512.x) Copyright © 2002 International Society of Nephrology Terms and Conditions