After rinsing twice with PBS, cells were fixed for 30 min in 4% paraformaldehyde, permeabilized using 0.2% Triton X-100, and blocked using 0.5% bovine serum albumin (BSA) at room temperature. Thereafter, cells were probed overnight with anti-DNMT1 (#ab188453), anti-VDR (#ab89626), anti-PTEN (#ab137337) (Abcam, Cambridge, MA, UK), anti-DNMT1 (#sc-271,729), anti-SNAI1 (#sc-271,977), anti-nephrin (#sc-376,522) (Santa Cruz, CA, USA), anti-E-cadherin (#14,472), and anti-p-NF-κB P65 (#3033) (Cell Signaling Technology, Danvers, MA, USA), anti-HBx (#MA1-81021) (Thermo Fisher Scientific, Waltham, MA, USA) antibodies at 4 °C. This was followed by a 60 min incubation with fluorescence-conjugated secondary antibodies (Yeasen, Shanghai, China) in the dark at room temperature. Then, cells were stained with DAPI (Beyotime, Shanghai, China) for 10 min at room temperature.
Anti vdr
Manufactured by Abcam
Sourced in United States
Anti-VDR is a protein-specific antibody that recognizes the vitamin D receptor (VDR) protein. VDR is a nuclear receptor that mediates the physiological effects of vitamin D. This antibody can be used in various laboratory applications, such as Western blotting, immunohistochemistry, and immunoprecipitation, to detect and study the VDR protein.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Anti pten, by Abcam (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti nephrin, by Santa Cruz Biotechnology (1 mentions)
Anti dnmt1, by Abcam (1 mentions)
Anti dnmt1, by Santa Cruz Biotechnology (1 mentions)
Anti snai1, by Santa Cruz Biotechnology (1 mentions)
Anti p nf κb p65, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Anti p70s6k, by Abcam (1 mentions)
Anti tsc2, by Cell Signaling Technology (1 mentions)
Anti rheb, by Cell Signaling Technology (1 mentions)
Anti 4e bp1, by Abcam (1 mentions)
Anti tsc1, by Cell Signaling Technology (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Anti mtor, by Abcam (1 mentions)
Anti pparγ, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Sangon (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
Horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
6 protocols using anti vdr
1
Immunofluorescence Assay of Cellular Markers
2
Western Blot Analysis of mTOR Pathway
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Proteins were extracted from RMCs using RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mM PMSF, and protease cocktail at 1 μg/mL). Protein concentrations were determined using a BCA kit (Thermo Scientific, Rockford, AL, USA). Equal amounts of protein (60 µg) were separated by SDS-PAGE on a 6%, 10%, or 12% acrylamide gel and then transferred onto a nitrocellulose membrane. After the membrane had been blocked with non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 for 1 h at room temperature, it was probed with the following primary antibodies overnight at 4°C: anti-VDR (catalogue no.: ab109234, Abcam, USA), anti-DDIT4 (catalogue no.: NBP1–77321, Novus Biologicals, USA), anti-TSC1 (catalogue no.: 6935S, Cell Signaling Technology, USA), anti-TSC2 (catalogue no.: 4308P, Cell Signaling Technology), anti-Rheb (catalogue no.: 13879S, Cell Signaling Technology), anti-mTOR (catalogue no.: Ab51044, Abcam), anti-4E-BP1 (catalogue no.: ab2606, Abcam), and anti-p70S6K (catalogue no.: ab32359, Abcam). After extensive washing, the membranes were incubated with Dylight anti-rabbit IgG secondary antibody. The antigens were visualized using the Odyssey infrared imaging system (LI-COR Biotechnology, Nebraska, USA). The results were expressed as the relative intensity (RI) intensity (adjusted to that of β-actin) of each band.
3
Molecular Mechanisms in Diabetic Kidney Disease
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Streptozotocin (STZ) was purchased from Sigma‐Aldrich (St. Louis, Missouri, USA). The following rabbit anti‐rat polyclonal antibodies were used: anti‐VDR (Abcam, Cambridge, Massachusetts, USA), anti‐PPAR‐γ (Santa Cruz Biotechnology, Santa Cruz, California, USA), anti‐transforming growth factor beta 1 (TGF‐β1), anti‐nuclear factor kappa B (NF‐κB), anti‐phospho‐NF‐κB, anti‐B‐cell lymphoma 2 (Bioworld Technology, St. Louis Park, Minnesota, USA), anti‐Bcl‐2‐associated X protein (Bax), anti‐inhibitor of NF‐κB alpha and anti‐β‐actin (Sangon Biotech, Shanghai, China).
4
Vitamin D Receptor Localization
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Pigmented and non-pigmented B16-F10 cells were treated for 24 h with 10 µM 1,25(OH)2D3, calcipotriol ((1S,3R,5Z,7E,22E,24S)-26,27-Cyclo-9,10-secocholesta-5,7,10,22-tetraene-1,3,24-triol) or 21(OH)pD. The Nuclear Extract Kit (Active Motif, La Hulpe, Belgium) was used for preparation of nuclear and cytoplasmic fractions. The protein concentration was measured by the Bradford method and samples were stored at −80 °C. The detection of VDR, PDIA3 and actin in nuclear and cytoplasmic extracts was performed using previously described methodology (46). Anti-VDR (1:100, Abcam, Cambridge, Great Britain), anti-PDIA3 (1:250, Sigma-Aldrich, Poznan, Poland) and anti-AKTB (1:200, Santa Cruz Biotechnology, CA, USA) antibodies were used to detect the antigens. The appropriate secondary antibody coupled to horseradish peroxidase (1:2000, Santa Cruz Biotechnology, Heidelberg, Germany) was then applied and proteins were visualized using Super Signal West Pico (Pierce Biotechnology, Rockford, IL, USA).
5
Western Blot Analysis of PEDV Signaling
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After washing with PBS, the cells were acquired by RIPA lysis buffer with PMSF and phosphatase inhibitor. Then, the cell samples were homogenized and centrifuged at 4 °C. The supernatants were collected. Then, the samples were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked by 5% nonfat milk. After this, the membranes were incubated overnight with the corresponding antibodies: anti-PEDV (Medgene Labs, Brookings, SD, USA); anti-STAT1, anti-p-STAT1, anti-STAT3, anti-p-STAT3, anti-JAK1, anti-JAK2, anti-NF-κB, anti-p-NF-κB, anti-IκBα (Cell Signaling Technology, Shanghai, China); anti-VDR (Abcam, Shanghai, China); anti-SOCS3 and anti-β-actin (Santa Cruz, Shanghai, China). Following washing, the samples were incubated with secondary antibodies for 1 h at room temperature, and then proteins were incubated with ECL reagent (Beyotime Biotechnology, Shanghai, China) for chemiluminescence by the ChemiDocTM XRS Imager System (Bio-Rad, Hercules, CA, USA).
6
Immunohistochemical Analysis of Vitamin D Pathway
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Immunohistochemistry was performed as previously described (21 (link)). Briefly, paraffin-embedded tumor specimens were subjected to a heat pretreatment at 60 °C for 1 hour, dewaxed in xylene, rehydrated in a series of ethanol, and treated with 0.01 mol/L citrate buffer (pH 6.0) for antigen retrieval. After inhibition of endogenous peroxidase activity for 30 minutes with methanol containing 0.3% H2O2, the sections were stained with anti-VDR (1:200, Abcam), anti-CYP27B1 (1:200, Abcam), or anti-CYP24A1 antibody at 4 °C overnight, followed by incubation by horseradish peroxidase (HRP)-labeled secondary antibody. The staining intensity was scored from 0 to 3 (0 = negative, 1 = mild staining, 2 = moderate staining, and 3 = strong staining) for every cell. An H-score is given using the following formula: [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)]. The final score was within 0-300 to quantify the protein expression.
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