1. Volume 71, Issue 11, Pages 1155-1162 (June 2007)
R990G polymorphism of calcium-sensing receptor does produce a gain-of-function and predispose to primary hypercalciuria 
G. Vezzoli, A. Terranegra, T. Arcidiacono, R. Biasion, D. Coviello, M.L. Syren, V. Paloschi, S. Giannini, G. Mignogna, A. Rubinacci, A. Ferraretto, D. Cusi, G. Bianchi, L. Soldati 
Kidney International 
Volume 71, Issue 11, Pages (June 2007)
DOI: /sj.ki
Copyright © 2007 International Society of Nephrology Terms and Conditions

2. Figure 1
(a) Distribution of variant alleles in hypercalciuric (n=124) and normocalciuric women (n=119). The 990G allele was more frequent in hypercalciuric than normocalciuric women. The distribution of the other variant alleles was not different in the two populations. Allele frequency is indicated at the top of the columns. (b) Frequency of four haplotypes in hypercalciuric and normocalciuric women. Haplotypes are explained in Table 1. Risk to be hypercalciuric was increased in women bearing haplotype GGC (characterized by variant allele only at the R990G SNP) and was calculated by logistic regression with haplotype GAC (women with no variant alleles) as reference group. Frequency of haplotype is indicated at the summit of each column. (c) Distribution of genotype at the R990G SNP in normocalciuric and hypercalciuric women. The distribution was different in hypercalciuric and normocalciuric women with an increased risk to be hypercalciuric in homozygotes or heterozygotes for the rarer 990G allele (n=23) compared with the homozygous women for the 990R allele (n=220).
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

3. Figure 2
[Ca2+]o-evoked increases in [Ca2+]i in HEK-293 cells transfected with the CaSR wild type (HEK-wt cells), or the 990G allele (HEK-990G cells), or empty vector (HEK-vector cells), loaded with fura-2AM. At each arrow, the concentration of [Ca2+]o was increased by adding a small volume of a CaCl2 stock solution to the bath solution. The gray double arrows indicate the peak of [Ca2+]i response after the individual [Ca2+]o stimulus in cells.23 The lower panel displays the tested concentrations of [Ca2+]o in the assay medium.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

4. Figure 3
Normalized transient [Ca2+]i response to stepwise [Ca2+]o increase. HEK-293 cells transfected with CaSR wild type, 986S, 990G, 1011E, or empty vector were loaded with fura-2AM. Error bar represents s.e. The transient [Ca2+]i response to the [Ca2+]o increase is plotted against the concentration of [Ca2+]o. Transient response was normalized to the maximal cumulative response of the CaSR. EC50 [Ca2+]o value in HEK-990G cells (2.69±0.11; n=19) was significantly lower than in HEK-wt (4.49±0.15; n=12; P=0.0001, analysis of variance with Sheffè test)), HEK-986S (4.28±0.25; n=11; P=0.0001), and HEK-1011E cells (4.04±0.25; n=9; P=0.0001). EC50 [Ca2+]o value was not different in HEK-wt, HEK-986S, and HEK-1011E cells.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

5. Figure 4
Immunoblot of CaSR in HEK-wt, HEK-986, HEK-990G, and HEK-vector cells. Total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose filter. After incubation with antihuman CaSR monoclonal antibody, filters were developed with enhanced chemiluminescence as described in the Material and Methods section. Upper panel shows three major bands of 250kDa (dimeric form), 130kDa (monomeric receptor), and 150kDa (glycosylated monomeric receptor). The same membrane was also incubated with anti-β-actin polyclonal antibody, as housekeeping expression control (lower panel).
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions