MA1-934

antibody from Invitrogen Antibodies

Targeting: CASR FHH, GPRC2A, HHC, HHC1, NSHPT

Provider product page for MA1-934

Western blot

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Immunohistochemistry

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Antibody data

Antibody Data
Antigen structure
References [54]
Comments [0]
Validations
Western blot [1]
Immunohistochemistry [4]
Other assay [49]

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Product number: MA1-934 - Provider product page
Provider: Invitrogen Antibodies
Product name: Calcium Sensing Receptor Monoclonal Antibody (5C10, ADD)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Reactivity: Human, Mouse, Rat, Bovine
Host: Mouse
Isotype: IgG
Antibody clone number: 5C10, ADD
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

CASR protein structure - MA1-934 shown in red.

Supportive validation

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Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized Human brain tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a mouse monoclonal antibody recognizing Calcium Sensing Receptor (Product # MA1-934) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized Human kidney tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a mouse monoclonal antibody recognizing Calcium Sensing Receptor (Product # MA1-934) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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Experimental details: Immunohistochemical analysis of mouse stomach stained with Calcium Sensing Receptor Monoclonal Antibody (Product # MA1-934). Fresh stomach tissue was fixed in 4% formalin for 1 hour and then incubated overnight at 4°C in 25% sucrose before embedding in tissue freezing medium. Antigen retrieval was carried out on 8 µm cryo-sections by incubating in sodium citrate buffer for 45 minutes at 4°C, immersing in sodium citrate buffer for 10 minutes at 100°C before washing 3 times for 5 minutes each in 1X PBS. Sections were then blocked in blocking buffer (0.3% Triton X-100 in 1X PBS containing 10% normal goat serum) for 30 minutes at RT before staining with Product # MA1-934 (diluted 1:100 in blocking buffer) overnight at 4°C followed by a fluorophore-conjugated goat anti-mouse IgG secondary antibody for 2 hours at RT. Sections were also stained with DAPI nuclear stain (blue). Product # MA1-934 positive cells (green) were found at the base of the antral glands in the mouse stomach. Data courtesy of the Innovators Program.

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Experimental details: Immunofluorescent analysis of bovine corneal epithelium (CE) limbus tissue extracts using Calcium Sensing Receptor Monoclonal Antibody (Product # MA1-934).

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Experimental details: Figure 6. Impact of Ca 2+ o concentration on phosphorylation of CaSR residue T888. (A) Detection and quantitation of CaSR T888 phosphorylation, and total CaSR in HEK-CaSR cells that were incubated in 0.2% BSA supplemented DMEM at 0.5, 3.0, or 5.0mM Ca 2+ o for 24 hours, followed by Western blotting. Over the extended period of the experiment, we did not observe high Ca 2+ o -induced dephosphorylation of CaSR-T888. Representative Western blot from 6 independent experiments. (B) Quantitation of Western blot intensity data. In response to either 3.0mM or 5.0mM Ca 2+ o a stable increase in pCaSR T888 was observed for the 140 kDa (high mannose) form but not the 160 kDa (complex glycosylated) form.

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Experimental details: Figure 1 CaSR protein expression in HUVECs by Immunofluorescence Confocal Microscopy and Western Blot. Immunofluorescent localization of CaSR in HUVECs with specific antibody and negative control after fixation and permeabilization protocol (A and B), or after fixation but not membrane permeabilization (C and D). Representative Western Blot of CaSR protein levels in HAoVSMC, HAEC and HUVEC total lysate, and in HUVEC membrane and cytoplasm extracts (E).

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Experimental details: Figure 5 Visualization of CaSR in murine, porcine, and human G-cells . Double-immunolabeling of horizontal tissue sections through the antral mucosa of mouse, swine and men for CaSR (green), and gastrin (red). (A,D,G) The CaSR antibody labels numerous cells in the antrum mucosa of mouse (A) , pig (D) , and men (G) . (B,E,H) Gastrin-positive cells are located in the same antral regions. (C,F,I) Overlay of accordant images; murine (C) , porcine (F) , and human (I) CaSR-positive cells are also immunoreactive for gastrin. Sections are counterstained with DAPI (blue). Scale bars: (A-I) = 20 mum.

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Experimental details: Figure 7 A small subpopulation of CaSR-positive cells contains somatostatin (Sst) . Double-label immunohistochemistry employing antibodies for CaSR (green) and Sst (red) on horizontal tissue sections through the antrum of mouse, pig, and men. (A,D,G) The CaSR antibody labels numerous cells in the antrum mucosa of mouse (A) , pig (D) , and men (G) . (B,E,H) In the same annuli numerous Sst-positive cells can be visualized. (C,F,I) Overlay of accordant images; subpopulations of murine (C) , porcine (F) , and human (I) CaSR-positive cells show immunoreactivity for somatostatin. Sections are counterstained with DAPI (blue). Scale bars: (A-I) = 20 mum.

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Experimental details: Figure 3 Serotonin deficiency decreases calcium transporters expression in the mammary gland on d 10 of lactation. Wild-type (WT) mouse dams were given saline and Tryptophan hydroxylase knock-out dams given either saline ( Tph1 -/- ) or daily injections of exogenous 5-HTP (100 mg/kg; Tph1 -/- (5-HTP)), from pregnancy d20 to lactation d10. Mammary gland mRNA and protein expression of calcium sensing receptor (CaSR, A1-2), calcium release-activated calcium channel protein 1 (ORAI-1, B1-2) and plasma membrane Ca 2+ ATPase and 2 (PMCA2, C1-2) on d10 of lactation. (D) Mammary mRNA expression of sodium/calcium exchanger 1 (NCX1), sarcoplasmic endoplasmic Ca 2+ ATPase 2 (SERCA2), plasma membrane Ca 2+ ATPase 1 (PMCA1), secretory pathway Ca 2+ ATPase 1 and 2 (SPCA1 and 2) on d10 of lactation. All values are the mean +- SEM (n = 7). Groups with different letters are significantly different ( P

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Experimental details: 1 Expression of calcium-sensing receptor (CaSR) in rat vagal bronchopulmonary sensory neurons, trachea and lung parenchyma A , CaSR immunostaining (green) in a cryosection of nodose ganglion. B , negative control with primary antiserum for CaSR substituted by its isotype, normal mouse IgG2a, in a nodose section. C , negative control with primary antiserum (Ab) for CaSR omitted in a nodose section. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) labelling (red) shown in the middle and right panels in A - C identifies the sensory neurons innervating the airway and lung structures. D , left panel, CaSR staining in trachea; middle panel, isotype control for CaSR in trachea; and right panel, CaSR staining in lung parenchyma. All tissues were sectioned at 12 mum; scale bars represent 50 mum.

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Experimental details: Figure 5 ( A ) Western Blot (standard reducing condition) for CaSR protein in hSC lines and during myogenic differentiation. The results show the presence of faint bands of about 160 kDa and 140 kDa in hSC lines and at different time points (T 0 , 3, 6, 9 days of myogenic differentiation) of primary hSC lines. HEK CaSR were used as positive control. Experiments were carried out in triplicate and are representative of the three established hSC lines. ( B ) Western blot analysis of CaSR in reducing and non-reducing conditions. The analysis for CaSR protein in three hSC lines showed that the observed bands are not CaSR as they did not shift to dimer form at 240 kDa in non-reducing conditions. HEK CaSR were used as positive control. The red and yellow boxes surround the analyzed bands.

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Experimental details: Figure 6 Human skeletal muscle tissue (hSMt) sections; observations in phase contrast microscopy, original. Magnification: 10x, scale bar 200 um ( A - C ). Immunostaining of CaSR protein. Analysis of CaSR protein in human skeletal muscle tissue sections: ( D ) negative control only with secondary antibody, ( E ) control with anti-CaSR antibody absorbed with blocking peptide, and ( F ) with primary anti-CaSR antibody. Analysis of CaSR protein in human parathyroid tissue sections used as positive control: ( G ) negative control with only secondary antibody, ( H ) control with anti-CaSR antibody absorbed with blocking peptide, and ( I ) with primary anti-CaSR antibody. Fluorescent microscopy in conventional colors: red for CaSR and blue for nuclei, original magnification: 10x, scale bar: 200 um. Experiment was carried out in triplicate (in hSMt sections of three different humans).

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Experimental details: Figure 7 Immunostaining of CaSR in hSC lines. The analysis of CaSR protein in hSC lines: ( A ) negative control with only secondary antibody and ( B ) with primary anti-CaSR antibody. Results are representative of experiments carried out in three established hSC lines. The analysis of CaSR protein was also performed in human parathyroid cells, used as positive control, ( C ) negative control with only secondary antibody, and ( D ) with primary anti-CaSR antibody. LSCM conventional colors: green for CaSR protein and red for nuclei, original magnification: 20x, scale bar: 50 um.

Supportive validation

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Experimental details: Figure 6. Impact of Ca 2+ o concentration on phosphorylation of CaSR residue T888. (A) Detection and quantitation of CaSR T888 phosphorylation, and total CaSR in HEK-CaSR cells that were incubated in 0.2% BSA supplemented DMEM at 0.5, 3.0, or 5.0mM Ca 2+ o for 24 hours, followed by Western blotting. Over the extended period of the experiment, we did not observe high Ca 2+ o -induced dephosphorylation of CaSR-T888. Representative Western blot from 6 independent experiments. (B) Quantitation of Western blot intensity data. In response to either 3.0mM or 5.0mM Ca 2+ o a stable increase in pCaSR T888 was observed for the 140 kDa (high mannose) form but not the 160 kDa (complex glycosylated) form.

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Experimental details: Figure 1 The expression and location of calcium-sensing receptor (CaSR) in bladder. ( a ) Western blot analysis of CaSR (MW: 130 kDa) in the whole kidney and whole urinary bladder. ( b ) Immunohistochemistry staining of CaSR (brown signals) in the urothelium (U) and detrusor (D) of bladder, and in the proximal tubule (PT) of kidney, and the staining without applying primary antibody (Ab). ( c ) Immunofluorescence staining of CaSR (red), uroplakin III A (UPK3A) (green), and DAPI (blue) in urothelium of urinary bladder.

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Experimental details: Figure 2 Establishment of the PTH-secretion potential of T-MSC-PTHCs from three donors: ( A ) conditioned medium was taken each step of T-MSC and T-MSC-PTHCs, and the concentration of PTH was measured using a commercial PTH ELISA kit at high cell density. Data are presented as the mean +- SD of at least three experiments (* p < 0.05, ** p < 0.01). PTH concentrations were significantly increased in T-MSC-PTHCs, compared with T-MSCs derived from donors #1 and #2, respectively. The expression of CaSR protein during differentiation of T-MSCs into T-MSC-PTHCs was measured by Western blotting and quantified using ImageJ software at low ( B ) and high ( C ) cell densities. Protein levels are normalized to GAPDH. Data are presented as the mean +- SD of at least three experiments (*** p < 0.001). CaSR expression levels were significantly increased in T-MSC-PTHCs, compared with T-MSCs derived from donors #1 and #3, respectively. Abbreviations: PTH, parathyroid hormone; T-MSC, tonsil-derived mesenchymal stem cells; T-MSC-PTHCs, T-MSC-derived PTH-releasing cells; CaSR, calcium-sensing receptor: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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Experimental details: Figure 3 Comparison of differentiation potential of T-MSC-PTHCs according to cell density during differentiation (low cell density: A, B; high cell density: ( C , D )): ( A , C ) the expression of PTH (green) in T-MSC (panel a) and T-MSC-PTHCs (panel b) derived from donor #1 was evaluated by immunostaining. The cells were counterstained with DAPI (blue). The expression of PTH of T-MSC-PTHCs was higher in high-density cells ( C ) than low-density cells ( A ); ( B , D ) expression of CaSR protein according to extracellular calcium level in T-MSC-PTHCs from three donor #1. T-MSC-PTHCs were further exposed to culture media containing low (0.09 mM) or high (3.0 mM) calcium concentrations for up to 48 h. The expression of CaSR was measured by Western blotting and quantified using ImageJ software and are normalized to GAPDH. Data are presented as the mean +- SD of at least three experiments (** p < 0.01, **** p < 0.0001). In both densities of cells, the expression of CaSR protein was significantly increased under exposure to low calcium and was not significantly increased under exposure to high calcium. Abbreviations: PTH, parathyroid hormone; T-MSC, tonsil-derived mesenchymal stem cells; T-MSC-PTHCs, T-MSC-derived PTH-releasing cells; CaSR, calcium-sensing receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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Experimental details: Figure 6. Immunolocalization and identification of calcium-sensing receptor (CaSR) protein and mRNA. a, Amplicons of the anticipated size (283 bp) were produced by RT-PCR amplification of RNA obtained from rat mesenteric artery (rma) and kidney (rk) and from porcine coronary artery endothelial cells (pca E). b, Western blot analysis of homogenates obtained from HEK293 cells transfected with CaSR (HEK), rat mesenteric and porcine coronary arteries (pca), and porcine coronary artery endothelial cell samples (each 35 &mgrg protein) using mouse monoclonal anti-CaSR antibodies. c and d, Same transverse section of rat mesenteric artery without (c) or with (d) DAPI staining of nuclei (blue). Immunoreactivity to the anti-CaSR antibody (red) was observed in the single layer of endothelial cells which is separated from the multiple layers of myocytes by the internal elastic lamina (green autofluorescence). The external elastic lamina defines the inner limit of the adventitial layer in which strong immunoreactivity to the antibody was also observed. e, Section of artery incubated with DAPI and secondary, but not primary, antibody. In (c through e), the horizontal scale bar represents 100 &mgrm. After density gradient separation of membrane fractions from (f) rat mesenteric artery and (g) porcine coronary artery endothelial cells, SK3 immunoreactivity was associated with the caveolin-1-rich fraction (C) whereas IK1 and CaSR were predominantly associated with the less buoyant (NC2) of the

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Experimental details: Figure 2. Upregulation of CaSR in PASMC from IPAH patients. A , Western blot analysis on CaSR in total proteins ( top ) and membrane proteins ( bottom ) isolated from normal PASMC (Nor) and IPAH-PASMC (IPAH). B , Summarized data (mean+-SE), bottom showing CaSR protein levels that were normalized to the &bgr-tubulin level. &ast&ast P

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Experimental details: Figure 4. Downregulation of CaSR with siRNA attenuates extracellular Ca 2&plus -induced &lsqbCa 2&plus &rsqb cyt increases in IPAH-PASMC and inhibits IPAH-PASMC proliferation. A , Western blot analysis on CaSR in IPAH-PASMC treated with a control (or scrambled) siRNA (control) and siRNA for CaSR (at 25 and 50 nmol&solL, n=3 for each group). B , Representative records of &lsqbCa 2&plus &rsqb cyt changes ( left ) and summarized data (mean+-SE) ( right ) showing the extracellular Ca 2&plus -induced &lsqbCa 2&plus &rsqb cyt increases in IPAH-PASMC transfected with control siRNA (n=57) and CaSR-siRNA (n=57). &ast&ast P

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Experimental details: Figure 5. Overexpression of CaSR in normal PASMC enhances extracellular Ca 2&plus -induced &lsqbCa 2&plus &rsqb cyt increase and promotes cell proliferation. A and B , Representative records of &lsqbCa 2&plus &rsqb cyt changes ( left ) and summarized data (mean+-SE) ( right ) extracellular Ca 2&plus -induced &lsqbCa 2&plus &rsqb cyt increases in human ( A ) and rat ( B ) PASMC transfected with an empty vector (vector, n=12) and the human CaSR cDNA (CaSR, n=8). C , Summarized data (mean+-SE) showing the total numbers of normal human PASMC transfected with the vector (solid circles) and CaSR gene (gray circles) after incubation in growth media for 24, 48, 72, 96, and 120 hours. The growth curves for vector control PASMC and IPAH PASMC are significantly different ( P

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Experimental details: Figure 6. Upregulation of CaSR in PASMC from rats with MCT-induced pulmonary hypertension. A and B , Regular ( A ) and real-timer ( B ) RT-PCR analyses on CaSR in PASMC isolated from normal rats (Norm, n=6) and rats with MCT-induced pulmonary hypertension (MCT, n=5). &ast&ast P

Supportive validation

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Experimental details: Figure 8. Upregulation of CaSR in PASMC from HPH mice and blockade of CaSR by NPS 2143 inhibits the development of HPH in mice. A and B , Real-time RT-PCR ( A ) and Western blot ( B ) analyses on CaSR in PA and lung tissues isolated from normoxic (Nor) and hypoxic (Hyp) mice. C , Representative record ( left ) and summarized data (mean+-SE, right ) showing the extracellular Ca 2&plus -induced increase in &lsqbCa 2&plus &rsqb cyt in PASMC isolated from normoxic and hypoxic mice. &ast&ast P

Supportive validation

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Experimental details: Figure 1. CYP11B1, CYP11B2, PTH1R, CaSR, and VD3R immunohistochemical expressions of irregular cord-like distribution in case 1.

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Experimental details: Figure 2. CYP11B1, CYP11B2, PTH1R, CaSR, and VD3R immunohistochemical expressions of multiple germinal centers in case 2.

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Experimental details: Figure 5. CYP11B1, CYP11B2, PTH1R, CaSR, and VD3R multiple fluorescence immunohistochemistry with overlapping distribution areas in case 2.

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Experimental details: Figure 5 Effect of Phe (0 mM, 50 mM, and 80 mM) on the levels of CaSR-mediated downstream pathway in the pig duodenum. The perfusion of duodenal tissues were done with 0 (control group), 50 mM, and 80 mM Phe for 160 min, respectively. During the perfusion, the perfused tissues were recovered every 10 min, starting from 40 min of perfusion to 110 min of perfusion, to extract total protein. ( a , e ) The measurement of protein levels of ( b , f ) CaSR, ( c , g ) PKC, and ( d , h ) IP 3 R in the duodenum tissues was performed by western blot analysis, using beta-actin as a loading control. Data are expressed as mean +- SEM ( n = 3). Dissimilar letters represent a significant difference at p < 0.05.

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Experimental details: Figure 6 Effect of the CaSR antagonist NPS 2143 on the CaSR-mediated downstream pathway in the presence of 80 mM Phe in pig duodenum. The tissues were perfused with 80 mM Phe and 25 uM NPS 2143 (specific CaSR antagonist) for 160 min. During the perfusion, the perfused tissues were recovered every 10 min, starting from 50 min to 120 min, to extract total protein. ( a ) The measurement of protein levels of ( b ) CaSR, ( c ) PKC, and ( d ) IP 3 R in the duodenum tissues was performed by western blot analysis, using beta-actin as a loading control. Data are expressed as mean +- SEM ( n = 3). Dissimilar letters represent a significant difference at p < 0.05.

Supportive validation

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Experimental details: Figure 1. The intracellular PTH and CaSR proteins were evaluated with immunofluorescence staining in adenoma parathyroid cells after 5 days in culture with growth medium and then transferred into glass slides and incubated for 24 h in DMEM medium containing 1.2 mM calcium before the phase contrast analysis and the immunofluorescence staining. (A) Phase contrast image of an adenoma parathyroid primary culture (40x original magnification). (B) Parathyroid cells stained for CaSR (in green) and counterstained for nuclei (in red). Observations with laser scanning confocal microscopy, objective 40x original magnification. (C) Parathyroid cells stained for PTH (in green) and counterstained for nuclei (in red). Observations with laser scanning confocal microscopy, 40x original magnification.