Journal: American journal of physiology. Cell physiology

Article Title: Notch enhances Ca 2+ entry by activating calcium-sensing receptors and inhibiting voltage-gated K + channels.

doi: 10.1152/ajpcell.00487.2019

Figure Lengend Snippet: Fig. 6. Notch activation or calcium-sensing receptor (CaSR) activation induces clustering of stromal interaction molecule 2 (STIM2) in CASR/STIM2- cotransfected human embryonic kidney 293 (HEK293) cells. A: representative fluorescent images (a) showing STIM2 clusters (green) in HEK293 cells transfected with CASR/YFP-STIM2 construct before (control) and during the treatment with cyclopiazonic acid (CPA). Magnified images (b) showing STIM2 clusters (arrows) in the puncta area and 3-dimensional images (c) showing STIM2 clusters in the puncta area as indicated in the magnified images. B: line scan graph (left) showing distribution and fluorescent intensity of STIM2 along the white dashed lines of the region indicated in b. Summarized data (means SE; right) showing the changes in fluorescent density of yellow fluorescent protein (YFP)-STIM2 before (control) and during application of CPA in CASR/STIM2- cotransfected HEK293 cells; n 5 independent sets of transfected cells. C: representative fluorescent images (a) showing STIM2 (green) in cells transfected with CASR/YFP-STIM2 before (control) and during the treatment with 50 M Jag-1 for 10 min in Ca2-free solution. Magnified images (b) showing STIM2 clusters (arrows) in the puncta area and 3-dimentional images (c) showing STIM2 clusters in the puncta area as indicated in the magnified images. D: line scan graph (left) showing distribution and fluorescent intensity of STIM2 along the white dashed lines of the region indicated in b. Summarized data (means SE; right) showing the changes in fluorescent density of YFP-STIM2 before and during application of Jag-1 in Ca2-free solution in CASR/STIM2-cotransfected HEK293 cells; n 6 independent sets of transfected cells. E: representative fluorescent images (a) showing STIM2 clusters (green) in HEK293 cells transfected with CASR/YFP-STIM2 before (0 Ca) and after 1.8 mM extracellular Ca2 treatment (Ca) for 10 min. Magnified images (b) showing STIM2 clusters (arrows) in the puncta area and 3-dimentional images (c) showing STIM2 clusters in the puncta area as indicated in the magnified images. F: line scan graph (left) showing distribution and fluorescent intensity of STIM2 along the white dashed lines of the region indicated in b. Summarized data (means SE; right) showing the changes in fluorescent density of YFP-STIM2 before and after 1.8 mM extracellular Ca2 treatment for 10 min in CASR/STIM2-co-transfected HEK293 cells; n 5 independent sets of transfected cells. We specifically selected regions of interest (ROIs) mostly close to the plasma membrane (i.e., the peripheral region of the cell) for analyzing the size, number, and intensity of STIM2 clusters. The summarized data (B, D, and F) show the overall intensity of the STIM2 clusters located in the peripheral regions of the cells. **P 0.01 and ***P 0.001 vs. control (Student’s t test).

Article Snippet: CASR construct was purchased from OriGene (Rockville, MD), yellow fluorescent protein (YFP)-STIM2 construct and green fluorescent protein (GFP) construct were purchased from Addgene (plasmid no. 18862, plasmid no. 19755), and KCNA5 construct was obtained from Harvard Medical School (plasmid HsCD00457881).

Techniques: Activation Assay, Transfection, Construct, Control, Clinical Proteomics, Membrane

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Relationship between CaSRs and LPS-injured cardiomyocytes

doi:

Figure Lengend Snippet: Expression of CaSR in cardiomyocytes as indicated by western blot (n=8). A basal level of CaSR was detected in the control group. This served as a baseline for the comparison of fold increases in other groups. The expression of CaSR in the LPS group was higher than that in the control group. Pretreatment with SC-211006 further increased CaSR expression. However, SC-207394 was found to inhibit the expression of CaSR. **P<0.05 vs. the control group, ▲▲P<0.05 vs. the LPS group.

Article Snippet: Reagents LPS was obtained from Sigma (St. Louis, USA), SC-211006 and SC-207394 were obtained from Santa Cruz Biotechnology, anti-CaSR antibody (product number: ACR-004) was purchased from Alomone Labs (Jerusalem, Israel), anti-GRP78 antibody (product number: 3158-1) was purchased from Epitomics (Burlingame, USA), and anti-LC3B antibody (product number: TDY357), anti-CytC antibody (product number: TDY378) and anti-Bcl-2 antibody (product number: TDY061) were obtained from Tian De Yue (Beijing, China).

Techniques: Expressing, Western Blot, Control, Comparison

Journal: FASEB BioAdvances

Article Title: Levo‐Tryptophan Promotes Osteogenesis Through Calcium‐Sensing Receptor

doi: 10.1096/fba.2025-00130

Figure Lengend Snippet: CaSR mediated the osteogenic‐promoting effects of L‐Trp on MC3T3‐E1 cells. (A) Expression levels of Casr and its downstream genes (Gna11 and Pth1r). At 4 days, Casr expression was significantly higher in the L‐Trp‐treated group compared to the control group. By 7 days, the expression of Gna11 and Pth1r was also significantly elevated in the L‐Trp‐treated group. (B, C) Qualitative and semi‐quantitative analysis ( n = 3) of CASR‐related proteins. L‐Trp significantly upregulated the expression of CASR and GNA11. (D, E) Cell scratching experiment and the semi‐quantitative analysis. L‐Trp significantly enhanced the migration of MC3T3‐E1 cells. This effect was reversed by NPS‐2143, a CASR inhibitor, indicating that L‐Trp promoted cell migration through CASR activation. (F, G) ALP staining and alizarin red staining. L‐Trp significantly enhanced osteogenesis and the effect was attenuated by NPS‐2143, confirming that L‐Trp‐mediated osteogenic differentiation through CASR activation.

Article Snippet: The primary antibodies included: CASR (1:500), GNA11 (1:1000), FAK (1:1000), RHOA (1:1000), Phospho‐FAK‐Y397 (1:2000), and Tubulin (1:5000) (All purchased from Abclonal, China).

Techniques: Expressing, Control, Migration, Activation Assay, Staining

Journal: The FASEB Journal

Article Title: Activation of the calcium sensing receptor increases claudin‐14 expression via a PLC ‐p38‐Sp1 pathway

doi: 10.1096/fj.202002137rrr

Figure Lengend Snippet: FIGURE 6 SP1 inhibits expression of the 1500 bp cldn14 promoter reporter construct. (A) Luciferase activity from the Cldn14 reporter construct after transfection with the transcription factor SP1. SP1 cotransfection significantly decreased luciferase activity of cells cotransfected with Empty Vector (EV) or the CaSR under low and high Ca2+ concentrations. Ordinary one-way ANOVA was used to determine statistical difference (***/###p < .001; ##p < .01; #p < .05). (B) Immunoblots demonstrating co-expression of the CaSR with SP1. (C) Immunohistochemical staining of a murine renal section for SP1 (brown) and NKCC2 (blue). (D) high magnification image of thick ascending limb with nuclear localization of SP1. (E) Representative immunoblots of myc tagged SP1 run on regular or FeCl3-loaded gels. (F) Relative phospho-SP1 protein abundance decreased when cells were incubated in with high [Ca2+] and expressed the CaSR. Friedman test with Dunn's multiple comparisons test were used to determine statistical differences (*p < .05)

Article Snippet: CaSR mutants were created by performing site directed mutagenesis (Quick Change Lighting kit from Agilent, Santa Clara, CA, USA) on the human CASR construct in pCMV (Origene, Rockville, MD, USA) with specific primers listed in Table 4, according to the manufacturer's instructions.

Techniques: Expressing, Construct, Luciferase, Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Western Blot, Immunohistochemical staining, Staining, Quantitative Proteomics, Incubation