The Homer2/3 DKO mice were generously provided by Dr Paul F Worley (Johns Hopkins University School of Medicine, Baltimore, MD) (Huang et al. 2008 (link)) and all animal care and experimental procedures complied with institutional guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) in Yonsei University (IACUC approval no. 2014-0067). Primary cultured bone marrow-derived monocytes (BMMs) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and α-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and incubated in 5% CO2. To maintain BMMs, α-MEM was supplemented with 30 ng/mL M-CSF. Receptor activator of RANKL and M-CSF were purchased from KOMA Biotech (Seoul, Korea). Fura-2/AM was purchased from Teflabs (Austin, TX, USA). Pluronic F-127 was obtained from Molecular Probes. Anti-Homer2, anti-Homer3 and anti-NFATc1 antibodies were obtained from Santa Cruz Biotechnology. Anti-actin antibodies were purchased from Sigma-Aldrich.
Anti nfatc1 antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-NFATc1 antibody is a laboratory reagent used for the detection and analysis of the NFATc1 protein, a transcription factor involved in various cellular processes. This antibody can be used in techniques such as western blotting, immunohistochemistry, and immunofluorescence to identify and quantify the presence of NFATc1 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Anti actin antibody, by Merck Group (2 mentions)
Anti phospho iκb, by Cell Signaling Technology (2 mentions)
Recombinant m csf, by R&D Systems (2 mentions)
Rankl, by R&D Systems (2 mentions)
Anti gapdh antibody, by Merck Group (2 mentions)
Anti phospho akt antibody, by Cell Signaling Technology (2 mentions)
Anti akt antibody, by Cell Signaling Technology (2 mentions)
Anti trap antibody, by Santa Cruz Biotechnology (2 mentions)
Anti upar antibody, by Santa Cruz Biotechnology (2 mentions)
Horseradish peroxidase conjugated antibody to rabbit igg, by Cytiva (2 mentions)
Chip assay kit, by Cell Signaling Technology (2 mentions)
α minimum essential medium α mem, by Thermo Fisher Scientific (2 mentions)
Pluronic f 127, by Thermo Fisher Scientific (1 mentions)
M csf, by Koma Biotech (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Sudachitin, by Fujifilm (1 mentions)
Anti phospho sapk jnk, by Cell Signaling Technology (1 mentions)
Anti iκb, by Cell Signaling Technology (1 mentions)
Recombinant murine il 1β, by R&D Systems (1 mentions)
14 protocols using anti nfatc1 antibody
1
Generating Bone Marrow-Derived Monocytes
2
Osteoclastogenesis Regulation Protocol
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Sudachitin was obtained from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). LPS derived from Escherichia coli (O55:B5) was purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human M-CSF was kindly provided by the Morinaga Milk Industry Co. (Tokyo, Japan). Recombinant murine soluble RANKL (sRANKL) and recombinant murine IL-1β were obtained from R&D Systems (Minneapolis, MN) and PeproTech (Rocky Hill, NJ), respectively. Prostaglandin E2 (PGE2) was obtained from Sigma-Aldrich. The anti-phospho-Erk1/2, anti-Erk1/2, anti-phospho-SAPK/JNK, anti-SAPK/JNK, anti-phospho-IκB, and anti-IκB antibodies were purchased from Cell Signaling Technology (Danvers, MA). The anti-c-fos and anti-NFATc1 antibodies were obtained from Santa Cruz Biotechnology (San Diego, CA). The anti-cathepsin K, anti-DC-STAMP (clone 1A2) and anti-Atp6v0d2 antibodies were purchased from BioVision (Mountain View, CA), MILLIPORE (Temecula, CA) and AVIVA Systems Biology (San Diego, CA), respectively.
3
Protein Extraction and Western Blotting
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Cells were lysed on ice for 30 min with RIPA lysis buffer, which contains 50 mM Tris_HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS supplemented with protease inhibitors (10 mg/ml leupeptin, 10 mg/ml pepstatin A, and 10 mg/ml aprotinin). For western blotting, 25 μg of protein sample was resolved on 12.5% SDS-PAGE and electrotransferred onto nitrocellulose membranes (Whatman). The primary antibodies used were as follows: phospho-Akt, total Akt, phospo-p44/p42 ERK, total p44/p42 ERK, phospho-p38, total-p38, phospo-JNK, and total-JNK were purchased from Cell Signaling Technology (1:1000 dilution ratio). Anti-NFATC1 antibody was purchased from Santa Cruz (1:500 dilution ratio). β -actin or GAPDH was used as loading control. Horseradish peroxidase-conjugated secondary antibodies were used at a 1:5000 dilution. The antigen–antibody complexes were visualized using the enhanced chemiluminescence detection system (Millipore) following the manufacturer’s instructions. Immunoreactive bands were quantitatively analyzed in triplicate by normalizing the band intensities to their respective controls on scanned films using ImageJ software.
4
Mogrol Regulation of Osteoclastogenesis
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C57BL/6J mice were acquired from Xiamen University’s Study Animal Center, and all animal investigations were authorized by Xiamen University’s Animal Care and Use Committee (XMULAC20210037). Chengdu Must Biotechnology (Chengdu, China) have supplied mogrol with a purity >98%. Dimethylsulfoxide (DMSO) was used to dissolve mogrol. Biological Industries (BI, Beit Haemek, Israel) supplied alpha-modified Eagle’s medium (α-MEM) and penicillin–streptomycin solution. Gibco (Thermo Fisher Scientific, Waltham, United States) supplemented with fetal bovine serum (FBS). Recombinant M-CSF and RANKL were acquired from R&D Systems (Minneapolis, MN, United States). Cell Signaling Technology (Danvers, United States) provided antibodies (p-ERK, ERK, p-P38, P38, p-JNK, JNK, p-P65, P65, and IκBα). Antibodies against c-FOS and TRAF6 were obtained from Abcam (Cambridge, United Kingdom). Anti-NFATc1 antibody was acquired from Santa Cruz Biotechnology (CA, United States). Anti-GAPDH antibody was purchased from Proteintech (Wuhan, China). Dojindo (Kyushu Island, Japan) provided Cell Counting Kit-8 (CCK-8). Beyotime Biotechnology, Ltd. (Shanghai, China) provided Annexin V-FITC apoptosis kits.
5
Quantifying Osteoclast Differentiation
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BMMs were plated on glass coverslips and incubated in α-MEM containing 10% FBS, 10 ng/ml M-CSF, and 20 ng/ml RANKL, with or without 5 μM (-)-tubaic acid. After four days, the cells were fixed with 4% paraformaldehyde, treated with 0.25% Triton X-100, and stained with an anti-NFATc1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by an Alexa Fluor-488-conjugated secondary antibody. Rhodamine-conjugated phalloidin (Cytoskeleton, Denver, CO, USA) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used to stain F-actin and cell nuclei, respectively. Cells with actin rings or nuclear NFATc1 were counted from 10 random selected views using the Image J software (NIH, Bethesda, MA, USA).
6
Western Blot Analysis of Signaling Proteins
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Western blot analysis was performed as previously described 23 . Cells were washed twice with cold PBS, harvested, and then sonicated in lysis buffer containing 10 mM Tris–HCl buffer (pH 7.5), 1% SDS, 1% Triton X‐100, and a protease inhibitor cocktail (Roche, Mannheim, Germany). The protein concentration in each lysate was measured using a BCA protein assay kit (Pierce, Rockford, IL). Proteins in the supernatant were separated by electrophoresis on 10% SDS‐polyacrylamide gels and transferred to a PVDF membrane. We detected expressions of TRAP, NFATc1, IκBα, uPA, uPAR, GAPDH, phospho‐AMPK, AMPK, phospho‐Akt, Akt by using anti‐TRAP antibody (Santa Cruz Biotechnology, Dallas, TX), anti‐NFATc1 antibody (Santa Cruz Biotechnology), anti‐IκBα antibody (IMGENEX, San Diego, CA), anti‐uPA antibody (Santa Cruz Biotechnology), anti‐uPAR antibody (Santa Cruz Biotechnology), anti‐GAPDH antibody (Sigma–Aldrich), anti‐phospho‐AMPK antibody (Cell Signaling Technology, Danvers, MA), anti‐AMPK antibody (Cell Signaling Technology), anti‐phospho‐Akt antibody (Cell Signaling Technology), anti‐Akt antibody (Cell Signaling Technology) followed incubation with horseradish peroxidase‐conjugated antibody to rabbit IgG (Amersham Pharmacia Biotech, Uppsala, Sweden).
7
Western Blot Analysis of Cell Signaling
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Western blot analysis was performed as previously described 30 . We detected expressions of uPAR, TRAP, NFATc1, GAPDH, phospho‐Akt, or Akt by using anti‐uPAR antibody (Santa Cruz Biotechnology, Dallas, TX), anti‐TRAP antibody (Santa Cruz Biotechnology), anti‐NFATc1 antibody (Santa Cruz Biotechnology), anti‐GAPDH antibody (Sigma–Aldrich), anti‐phospho‐Akt antibody (Cell Signaling Technology, Danvers, MA) or anti‐Akt antibody (Cell Signaling Technology) followed incubation with horseradish peroxidase‐conjugated antibody to rabbit IgG (Amersham Pharmacia Biotech, Uppsala, Sweden).
8
Osteoclastogenesis Regulation by DTOGG
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Six-week-old C57/B6L mice were obtained from Dae Han Bio Link (Chungbuk, Korea). All animal experiments were approved by the committees on the care and use of animals in research at Kyungpook National University, and were conducted in accordance with the guidelines for the care and use of laboratory animals. Primary antibodies against phospho-p38, p38, phospho-JNK, phospho-ERK, ERK, phospho-p65, and phospho -IκBα were purchased from Cell Signaling Technology (Danvers, MA). Anti-NFATc1 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant M-CSF and RANKL were obtained from R&D Systems (Minneapolis, MN). Fetal bovine serum (FBS) and α-minimum essential medium (α-MEM) were purchased from Gibco BRL (Grand Island, NY). 2-O-digalloyl-1,3,4,6-tetra-O-galloyl-β-D-glucose (DTOGG, Fig. 1A ) was isolated from Galla Rhois collected in Asan-si, South Korea, and its extraction and isolation methods were described previously (10 (link)).
9
Murine Monocyte and Osteoblast Regulation
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DBA/1J mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). All animal experimental procedures were approved by the Animal Care and Use Committee of the National Defense Medical Center, Taiwan (IACUC:15-303). The murine monocyte RAW 264.7 cell line was purchased from the Food Industry Research and Development Institute, Taiwan. MC3T3-E1 was purchased from ATCC, USA. Fetal bovine serum, a-minimum essential medium (a-MEM), penicillin and streptomycin were purchased from Gibco (Grand Island, NY, USA) RANKL and M-CSF were purchased from Peprotech (London, UK). Antibody against TRAF-6 was obtained from Abcam (Cambridge, MA, USA) Anti-JNK, annti-ERK, anti-p38, anti-NF-κB p65, anti- IκBα, anti-phospho-ERK, anti-phospho-JNK, anti-phospho-p38, Anti-pospho-NF-κB p65, anti-phospho-IκB and anti-caspase-3 antibodies were all obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-NFATc1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Niclosamide, anti-ß-actin and anti-lamin B1 antibodies and all other reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
10
Immunofluorescence Analysis of Podocyte Markers
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Frozen kidney tissues were firstly cut into 4 μm thick sections and were incubated with anti-synaptopodin antibody (1:100, Santa Cruz, sc-21537), anti-podocin antibody (1:100, Sigma, P0372), anti-NFATc1 antibody (1:50, Santa Cruz, sc-13033) overnight at 4 °C, followed by incubation with fluorescein-conjugated secondary antibody. The results were examined with immunofluorescence microscopy (Leica DMLB, Wetzlar, Germany). For in vitro cultured podocytes, coverslips containing podocytes were firstly fixed with 4% paraformaldehyde and permeabilized in 0.3% Triton X-100 for 10 min. Podocytes were blocked with 10% FBS for 1 h and then incubated with FITC-phalloidin (1:100, Sigma, P5282), anti-synaptopodin, anti-podocin, and anti-NFATc1 antibody.
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