MA1-710

antibody from Invitrogen Antibodies

Targeting: VDR NR1I1, PPP1R163

Provider product page for MA1-710

Western blot

Immunocytochemistry

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Antibody data

Antibody Data
Antigen structure
References [114]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [2]
Other assay [37]

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Product number: MA1-710 - Provider product page
Provider: Invitrogen Antibodies
Product name: VDR Monoclonal Antibody (9A7)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MA1-710 detects vitamin D receptor (VDR) from human, rat, mouse, hamster, chicken, amphibian, and fish tissues. This antibody does not cross-react with estrogen or glucocorticoid receptors. MA1-710 detects both the occupied and unoccupied forms of the receptor.
Antibody clone number: 9A7
Concentration: 1 mg/mL

VDR protein structure - MA1-710 shown in red.

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Experimental details: Figure 7 Results of the in vivo study (A + B) and the primary cell culture (C + D) of Col2-pd2EGFP transgenic mice growth plate chondrocytes. A: Histomorphometry (H&E) and immunofluorescent staining for collagen type X (red) of tibial growth plates from Col2-Col2-pd2EGFP transgenic mice (6 weeks of age) indicating that vitamin D 3 deficient mice (VitD-) developed rickets. Supplementation with 1,25(OH) 2 D 3 (VitD-; +1,25D) reversed the rachitic phenotype as compared with controls (VitD+). Note that Col2-pd2EGFP fluorescence (green) is native (mean +- SD; n = 7). B: Relative ColX , Vdr , Pthrp , and Pthr1 gene expression corrected for reference genes in sorted Col2-pd2EGFP positive growth plate chondrocytes after termination of the study, at 6 weeks of age (mean +- SD; n = 6). Relative target gene expression in the growth plate of control (VitD+) mice was set at 1. C: Relative ColX , Vdr , Pthrp , and Pthr1 gene expression corrected for reference genes in control or 1,25(OH) 2 D 3 -treated cultured primary Col2-pd2EGFP growth plate chondrocytes. Time is shown in days after the start of differentiation (T 0 , T 3 , T 5 , and T 7 ). From T 2 onward, 10 -8 M 1,25(OH) 2 D 3 was daily added to the culture medium. Relative target gene expression at T 0 in the control culture was set at 1. These data were not subjected to statistical analysis due to the small sample size (n = 1 experimental replicate). D: From left to right: Immunofluorescent staining for collagen type X and native e

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Experimental details: Figure 3 Bladder epithelial cells are equipped with the machinery for local 25D 3 conversion and activity. Expression of CYP27B1, VDR and CYP24A1 was detected on mRNA level in normal bladder cells (HBEP) and three bladder cells lines (TERT-NHUC, 5637 and T24) by RT-PCR ( A , C , E ) and on protein level in tissue from bladder biopsies by fluorescence immunohistochemistry ( B , D , F ). mRNA expression results were normalized to expression in the kidney epithelial cell line A498. The proteins are stained with AlexaFluor 594 (red), the cell nucleus with DAPI (blue). CYP27B1 protein was detected in the bladder mucosa where it was evenly distributed in the bladder epithelial layers with an abundance of staining seen in the peri-nuclear area, consistent with its association to mitochondria ( B ). VDR protein was localized in the cytoplasm and nucleus ( D ). Low levels of the CYP24A1 protein were detected in bladder mucosa ( F ). All images are captured at x63 magnification and the right panels of images B, D and F show controls stained without primary antibodies against CYP27B1, VDR and CYP24A1, respectively.

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Experimental details: Figure 1 VDR and PTHrP expression in 1,25(OH) 2 D 3 - and PTHrP-treated ATDC5 chondrocytes in the prehypertrophic phase of differentiation. ATDC5 cells were treated starting from day 7 of differentiation (T 0 ) with 10 -8 M PTHrP or 1,25(OH) 2 D 3 at T 0 , T 24 , and T 48 . A. Relative Vdr gene expression (mean +- SD; n = 8). T 0 in the control culture was set at 1. B: VDR protein expression expressed as VDR/beta-actin ratio (mean +- SD; n = 6). T 0 in the control culture was set at 1. c = control culture, P = 10 -8 M PTHrP-treated culture, d = 10 -8 M 1,25(OH) 2 D 3 -treated culture. C: Relative Pthrp gene expression (mean +- SD; n = 8). T 0 in the control culture was set at 1. D: PTHrP protein levels corrected for DNA content (pg PTHrP/ng DNA) in ATDC5 culture media of control cultures and 10 -8 M 1,25(OH) 2 D 3 -treated cultures, determined using a PTHrP ELISA (mean +- SD; n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001.

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Experimental details: Figure 3 Identification of a functional vitamin D 3 response element (VDRE) in the PTHrP and PTHR1 promoter region. A: Schematic presentation of the mouse PTHrP promoter region with location of the vitamin D 3 response elements (VDREs). Primers were designed for VDRE1 and VDRE2. The location of VDRE1 and VDRE2 upstream from TSS is 913 and 571 bp, respectively. bp, base pairs; TSS, transcription start site. B: VDR ChIP assay performed on ATDC5 cells in the prehypertrophic phase of differentiation. Cells were fixed after 24 h of treatment with 10 -8 M 1,25(OH) 2 D 3 . IgG was used as normalization control and a sequence 1,000 bp upstream of VDRE2 was used as a negative control. Enrichment is given as mean +- SD; n = 8. The results indicate functional binding of 1,25(OH) 2 D 3 and its receptor, the VDR, to the PTHrP promoter region. C: Overview of expected VDREs in the PTHR1 promoter region. The PTHR1 gene has two promoter regions, P1 and P2. In bone and cartilage, the P2 promoter controls PTHR1 expression, whereas expression of P1 is restricted mainly to the kidney. The location of the expected VDRE1 (AGGTGA) and VDRE2 (GGTTGA) is 2,092; and 2,027 bp upstream from TSS, respectively. The arrows indicate the position of the primers used in this study. bp, base pairs; TSS, transcription start site. D: VDR ChIP assay performed on ATDC5 cells in the prehypertrophic phase of differentiation. The VDRE combination contains both the expected VDRE1 and VDRE2 sequence. The same conditions

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Experimental details: Figure 4 The role of VDR in the paracrine feedback loop between PTHrP and 1,25(OH) 2 D 3 . A,B: Relative Vdr and Pthrp gene expression in ATDC5 cells in the prehypertrophic phase of differentiation (mean +- SD; n = 6). In the VDR-oligo groups, silencing of the VDR was performed on differentiation day 4. At T 0 (day 7 of differentiation), T 24 , and T 48 , 10 -8 M 1,25(OH) 2 D 3 was added to the three 1,25(OH) 2 D 3 -treated culture groups. Relative target gene expression at T 0 in the control culture was set at 1. C: Western blot analysis confirming the successful siRNA transfection in 10 -8 M 1,25(OH) 2 D 3 -treated prehypertrophic ATDC5 cells with approximately 50% downregulation of VDR production. Time is shown in hours after 1,25(OH) 2 D 3 was first added and the VDR/beta-actin ratio (mean +- SD; n = 3) in the 1,25(OH) 2 D 3 culture at T 24 was set at 1. d = 10 -8 M 1,25(OH) 2 D 3 -supplemented culture, ds = 1,25(OH) 2 D 3 + scrambled mock culture, do = 1,25(OH) 2 D 3 + VDR-oligo culture. D: PTHrP protein levels (mean +- SD; n = 3) corrected for DNA content in ATDC5 culture media, determined using a PTHrP ELISA. * P < 0.05, ** P < 0.01, *** P < 0.001.

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Experimental details: Fig. 1 LSD1 expression in prostate tissues is increased in advanced prostate tumors. Western blot and immunohistochemistry (IHC) staining were used to measure protein levels of LSD1 and VDR in wild-type (WT) and TRAMP mice. CWR22 xenograft mice were used to investigate the role of LSD1 and VDR in PCa growth kinetics. a Western blotting image showing the expression of LSD1 and VDR protein levels in wild-type and TRAMP prostate lysates. WT wild-type mouse, T tumor/TRAMP mouse, CR castration-recurrent tumor from TRAMP mouse. b LSD1 ( left ) and VDR ( right ) protein quantification of LSD1, or VDR, normalized to GAPDH. Data from wild-type samples were compared with the data from tumor samples using Student's t test. p values are indicated in the plot. c LSD1 and VDR IHC staining in age-matched prostate samples of 25-week-old TRAMP and WT mice. Staining shows a strong nuclear localization, in brown , in both WT and TRAMP tumors, with a stronger signal in tumor. Labels in the image indicate protein (LSD1, VDR), magnification (x 10, x 20), and tissue type (WT, tumor (T)). d Kaplan-Meier plots showing time to recurrence for CWR22 xenografts, measured as time necessary for the tumor to reach 1000 mm 3 in volume. The X -axis indicates weeks of the experiment where time 0 is the time of testosterone pellet removal. The Y -axis indicates the percentage of mice with tumor that did not reach 1000 mm 3 . The black lines indicate mice with low LSD1/VDR levels, and the red lines indicate

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Experimental details: Figure 1 Expression of the VDR in aging mammary glands of WT mice. A. Real Time PCR for VDR gene expression in aging mammary glands (12, 14, and 16 months) derived from wild type (WT) mice were compared to mature 10 week (10wk) pubertal and late pregnancy mammary glands. Data are expressed relative to 18S RNA (Relative VDR Expression) and represent mean +- s.e.m. of triplicate runs. B. Western blot of VDR protein expression in aging mammary glands (12 and 14 months) from WT mice, which is undetectable in VDR KO glands. C. Formalin fixed sections of mammary gland from 12, 14, and 16 month old WT mice were subjected to immunohistochemistry with a monoclonal antibody directed against VDR. A representative stained section of a 12 month WT gland shows positive staining in the mammary epithelium, stroma and adipose, a pattern that is similar to pubertal mammary gland expression [15] . VDR positive cells appear brown against the blue hematoxylin counterstain.

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Experimental details: Figure 5 Representative western blot images (A) of chicken calcium-sensing receptor (cCaSR) and chicken vitamin D receptor (cVDR); the quantitative relative expression of cCaSR (B) and cVDR (C) in different tissues at the 40 wk of age (peak production). Data were presented as mean +- SEM, n = 6. Different letters represent a significant difference ( P < 0.05).

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Experimental details: Plate1 Immunohistochemical Staining of VDR and CTLA 4 in Breast Carcinoma Cases A: A case of IDC grade III exhibiting high cytoplasmic expression of CTLA 4 in tumor cells and positive expression in lymphocytes (IHC x 200), B: A case of IDC grade II exhibiting low cytoplasmic expression of CTLA 4 in tumor cells and positive expression in lymphocytes (IHC x 200), C: A case of IDC grade II exhibiting high nuclear expression of VDR (IHC x 200), D: A case of IDC grade II exhibiting low nuclear expression of VDR (IHC x 200). E: A case of IDC grade III exhibiting high nuclear expression of VDR (IHC x 200). F: A case of IDC grade I exhibiting low cytoplasmic expression of CTLA4 (IHC x 200).

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Experimental details: Figure 6 Impact of calcitriol intervention on disease phenotype and vitamin D signaling. The panel of images represent photomicrographs of hematoxylin and eosin stained tongue sections (H&E) along with matched fields of VDR and CYP24A1 (10X magnification). Corresponding Ki67 immunostained field (40X) magnification is also shown on the right. The images for the control (4NQO alone) and the three calcitriol regimens have been shown for normal tongue epithelium ( A ), low-grade dysplasia ( B ), high-grade dysplasia ( C ), and invasive cancer ( D ). Figure 6

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Experimental details: Expression of nuclear receptors for vitamin D3 hydroxyderivatives and CYP enzymes involved in vitamin D metabolism in murine ASZ001, human A431 and human SCC13 carcinoma lines. (A) Immunofluorescence detection (left panel) was performed using antibodies against VDR (MA1-710) and RORalpha and RORgamma (). The antigen is in green, while cell nuclei are red-counterstained with propidium iodide; scale bar=100 u m. Right panel shows detection of VDR and RORalpha and RORgamma by western blotting (arrows) using specific anti-receptor antibodies (see Materials and methods) for isolated cytoplasmic or nuclear fractions (VDR) from the cells or whole extracts (CYP27B1, RORalpha and RORgamma). Human hepatoma and human melanoma SKMEL-188 cells were used as positive controls for RORalpha and RORgamma. Loading was evaluated using antibodies against lamin C and alpha-tubulin for nuclear or cytoplasmic fractions, respectively, while with anti-beta-actin for whole extracts. (B) shows expression at the mRNA level of Cyp27b1, Cyp24a1, Cyp11a1, Vdr, Rora and Rorc , respectively in ASZ001 murine carcinoma line treated with 10 -7 M of the secosteroids listed on the x-axis. Data represent fold change, using beta-actin as a housekeeping gene and are shown as means +- SEM (n=4) with * P

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Experimental details: Figure 3 Protein and mRNA levels of HK2 cells analyzed by western blot and RT-PCR. (a), (b), and (c) show the protein levels of VDR, p-AKT, and UCP2 evaluated by western blot. (d) mRNA levels of VDR, AKT, and UCP2 measured by RT-PCR. Results are presented as the fold change in activity relative to normoxic cells. The data are expressed as the mean +- SD. *p

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Experimental details: Figure 5 Effects of calcitriol, PPAR stimulators and ERAD inhibitor on expression of VDR. ( A ) HCV-infected Huh7.5-SEAP cells (1.5 x 10 4 ) were treated with 5 muM of Wy14643, 10 muM of linoleic acid, 30 muM of Ly171883, or 20 muM of kifunensine for 1 h ahead of treatments with 100 nM of calcitriol for 6 days in a 24-well plate, fixed with 1% paraformaldehyde, stained with core and VDR antibodies and counterstained with DAPI. After staining, these cells were mounted on glass slides and observed under confocal laser scanning microscope. Scale bar, 10 muM; ( B ) The arbitrary units (AU) of immunofluorescence intensity were assessed using Image J; ( C ) Cell lysates (7 x 10 5 ) were collected and the VDR level was detected by Western blot. Data are expressed as mean +- SD obtained from three individual experiments. # p < 0.05 and ## p < 0.01 vs. compared with the HCV-infected Huh7.5-SEAP group. & p < 0.05 and && p < 0.01 vs. the calcitriol-treated group. The symbol (-) referred to the cells without HCV infection; the symbol (+) referred to the cells with HCV infection; the symbol (++) referred to the cells with HCV infection and co-treated with drug (e.g., calcitriol, individual PPAR agonist, or ERAD inhibitor); the symbol (+++) referred to the HCV-infected cells pre-incubated with individual PPAR agonist or ERAD inhibitor before calcitriol treatment.

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Experimental details: 2 Localization of VDR and 1alpha-OHase in human primary breast adipocytes. Adipocytes were plated and grown on chamber slides, formalin fixed, and either processed as unstained control cells (a,b) or stained to detect 1alpha-OHase (d), VDR (e), or the nuclear compartment using DAPI incorporation (c,f). Primary adipocytes were incubated with 1alpha-OHase (d) or without (a) primary antibody directed against 1alpha-OHase protein to assess the expression and localization within primary adipocytes. 1alpha-OHase expression is shown as brown cytoplasmic staining (d) against the blue hematoxylin counterstain (a,d). Immunofluorescent detection of VDR (e) shows nuclear staining within the adipocytes compared to the no primary antibody control (b). Nuclear incorporation of DAPI staining (c,f) of control cells (b) or VDR stained cells (f) emphasizes the nuclear localization of VDR expression.

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Experimental details: Fig. 5 BMP inhibition promotes osteoblast differentiation in myeloma in vivo via down-regulation of Wnt inhibitors. a Mineralisation of murine pre-osteoblast cell line 2T3 in the presence of LDN193189 or BMPR1a-FC. b - e Histomorphometric analysis of haematoxylin- and TRAP-stained femur sections from healthy and myeloma-bearing mice: n = 5 per control group, 10 per myeloma group, total n = 30. Missing data points: see Methods . b Bone-lining cell density (N.Ob/T.Ar, per mm 3 ) in mice treated with vehicle vs. LDN193189 and vehicle vs. BMPR1a-FC. c Mineral apposition rate (mum per day) and bone formation rate (mum 3 /mum 2 per day) in healthy and myeloma-bearing mice treated with vehicle vs. LDN193189. d Images of bone-lining cell layer in healthy mice treated with vehicle, LDN193189 or BMPR1a-FC. e Bone-lining cell layer thickness (mum) in healthy mice treated with vehicle vs. BMPR1a-FC and vehicle vs. LDN193189. f Images of bone-lining cell layer in healthy mice treated with vehicle vs. BMPR1a-Fc, stained for osterix and vitamin D receptor (VDR). Blue = Hoescht, green = osterix, red = VDR, yellow/orange = expression of osterix and VDR. Scale bar = 100 mum. g Gene sets enriched (FDR 25 genes) on GSEA of stromal progenitors from LDN193189 vs. vehicle-treated myeloma-bearing mice. h Effect of siRNA double knockdown of Acvr1 and Bmpr1a (rat osteosarcoma line UMR-106.01) on Acvr1 , Bmpr1a , Smad6 , Dkk1 and Sost (sclerostin) gene expression ( n = 4 replicates). Effect