case_images: PMC10858793_01
case_images: Western blot analysis of wild type and L34P variant CaSR examined under nonreducing and reducing conditions. HEK293 cells were transfected with wild type or variant CaSR-FLAG-tagged plasmid (5 microg) and then incubated for 48 hrs, and the cells were then lysed, and 80 microg protein, with or without a reducing agent, was separated by SDS-PAGE. The gel was blotted on to nitrocellulose and then probed with mouse monoclonal antibody to FLAG-tag (upper panel) and alpha-tubulin (as a loading standard; lower panel), followed by goat antimouse HRP-conjugated secondary antibody. Protein bands were visualized using ECL chemiluminescence reagent in a Bio-Rad Molecular Imager. Experimental details are described in reference 18. The image is representative of several independent experiments. The FLAG and tubulin immunodetections were derived from the same blot. Cropping was used to ensure a more concise presentation of the gel image (see Supplementary Figures 1 and 2).
case_images: CRIE2024-6652801.001.jpg
case_images: PMC10858793_01_CRIE2024-6652801.001.jpg
case_images: fig1
case_images: Supplementary Figures 1 and 2; supplementary figures represent uncropped immunoblots that comprise Figure 1). Since a mature, dimerized receptor is essential for CaSR signalling, these results suggested that the L34P variant would be devoid of signalling capacity; i.e., the receptor would be inactive.
case_images: Dose response curves for wild type, L34P variant, and a known inactivating (L174R) variant CaSR using the IP-One ELISA assay following Ca++ ion (1.5, 2.5, 4, 7, or 10 mM) stimulation in HEK293 cells. The IP-One ELISA assay was performed as described previously (see reference 18). Values represent the mean % stimulation (with +-2x standard error of the mean error bars) plotted against Ca++ ion concentration for three separate experiments. Calculation of % stimulation and statistical comparisons between the wild type and variant receptors were as described in reference 18.
case_images: CRIE2024-6652801.002.jpg
case_images: PMC10858793_01_CRIE2024-6652801.002.jpg
case_images: fig2
case_images: Supplementary Figures 1 and 2; supplementary figures represent uncropped immunoblots that comprise Figure 1). Since a mature, dimerized receptor is essential for CaSR signalling, these results suggested that the L34P variant would be devoid of signalling capacity; i.e., the receptor would be inactive. A typical dose response accumulation of D-myo-inositol-1-phosphate was observed for the wild type receptor; however, the L34P variant showed no significant response to Ca++ at any concentration of Ca++ ions (p < 0.01) and behaved similarly to a known inactivating receptor, L174R, which was used as a positive control for defective receptor function (Figure 2).