Nfatc1

Manufactured by Cell Signaling Technology

Sourced in United States, China

NFATc1 is a transcription factor that plays a key role in the regulation of gene expression in response to calcium signaling. It is involved in the activation of immune cells and the differentiation of various cell types.

Automatically generated - may contain errors

Lab products found in correlation

67 protocols using nfatc1

Protein Extraction and Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total protein was extracted from BMMs and C3H10 cells and from the bones of mice used in the OVX experiment using a radioimmunoprecipitation assay lysis buffer (RIPA) (Sigma-Aldrich), and the protein concentration was measured using a bicinchoninic acid protein (BCA) assay kit (EMD Millipore, Bedford, MA, USA). Protein equivalents were separated using 10% SDS PAGE and transferred onto PVDF membranes (EMD Millipore). The membranes were blocked for an hour at room temperature with 5% skim milk in Tris-buffered saline with Tween 20. After incubating primary antibodies overnight at 4 °C, the membranes were nurtured with corresponding HRP conjugated secondary antibodies (Proteintech Group, Chicago, IL, USA.) The bands were visualized employing ECL Luminous Liquid (EMD Millipore). ImageJ software was used to compute the relative grey level of proteins (NIH). Anti-TRAIL was procured from Santa Cruz Bio (Santa Cruz, CA, USA). Anti-CTSK, TRAP, NFATc1, OPG, RUNX2, OCN, RANKL, COL1a, p-P65, P65, p-P38, P38, p-JNK, JNK, p-ERK, ERK, p-IKBα, IKBα, IKKα, IKKβ, and p-IKKα/β were all purchased from Cell Signaling Technology (Danvers, MA, USA).

Li J., Li X., Zhou S., Wang Y., Lu Y., Wang Q, & Zhao F. (2022). Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL. Molecular Medicine, 28, 141.

+ Open protocol

+ Expand

Osteoclastogenesis Regulation by Stachydrine

Check if the same lab product or an alternative is used in the 5 most similar protocols

Stachydrine (purity, ≥98%) and SC79 (purity, ≥97%) were purchased from Selleck Chemicals. Alpha modification of Eagle's medium (α‐MEM), penicillin/streptomycin and foetal bovine serum (FBS) were obtained from Gibco‐BRL. Recombinant mouse M‐CSF and RANKL were purchased from R&D Systems. Primary antibodies against TAK1 (#5206), phosphorylated TAK1 (p‐TAK1) (#4508), IκBα (#4814), phosphorylated IκBα (p‐IκBα) (#2859), p65 (#8242), phosphorylated p65 (p‐p65) (#3033), IκB kinase β (IKKβ) (#8943), phosphorylated IKKα/β (p‐IKKα/β) (#2697), p38 (#9212), phosphorylated p38 (p‐p38) (#4511), ERK (#4695), phosphorylated ERK (p‐ERK) (#4370), JNK (#9252), phosphorylated JNK (p‐JNK) (#4668), phosphorylated Akt (p‐Akt) (#2965), Akt (#4685), phosphorylated GSK3β (p‐GSK3β) (#9323), GSK3β (#9315), NFATc1 (#8032), PCNA (#13110) and β‐tubulin (#2146) were obtained from Cell Signaling Technology. Primary antibodies against c‐Fos (ab208942), NFATc1 (ab2796) and CTSK (ab37259) were obtained from Abcam. Primary antibody against TRAP (sc‐376875) was obtained from Santa Cruz Biotechnology and against active p65 (MAB3026) was obtained from Millipore. Cell counting kit‐8 (CCK‐8) was obtained from Dojindo Molecular Technology. TRAP staining kit, DMSO, SC‐514 and other reagents were obtained from Sigma‐Aldrich, unless otherwise indicated.

Meng J., Zhou C., Zhang W., Wang W., He B., Hu B., Jiang G., Wang Y., Hong J., Li S., He J., Yan S, & Yan W. (2019). Stachydrine prevents LPS‐induced bone loss by inhibiting osteoclastogenesis via NF‐κB and Akt signalling. Journal of Cellular and Molecular Medicine, 23(10), 6730-6743.

+ Open protocol

+ Expand

Whole Cell Protein Extraction and Western Blotting

Check if the same lab product or an alternative is used in the 5 most similar protocols

For whole cell protein extracts, cells were collected and lysed with RIPA buffer (Boston Bioproducts, Ashland, MA). Protein was quantitated using the BCA assay (Pierce, Rockford, IL). Lysates were electrophoretically separated using 12% Tris HCl gels (Biorad, Hercules, CA) and transferred to nitrocellulose membranes. Western blotting was performed using antibodies specific for the following proteins: p STAT5, STAT5, pAkt, Akt, p56lck, phosphor-p56lck, p Erk1/2, Erk1/2, p FoxO1/FoxO3a, FoxO1, GATA-1 (all from Cell Signaling Technologies, Danvers, MA), NFATc1, goat anti-rabbit IgG-HRP, and goat anti-mouse IgG-HRP. Average densitometric ratio was calculated for each blot by normalizing to an actin loading control using Image J software.

Grishkan I.V., Tosi D.M., Bowman M.D., Harary M., Calabresi P.A, & Gocke A.R. (2015). Antigenic stimulation of Kv1.3-deficient helper T cells gives rise to a population of Foxp3-independent T cells with suppressive properties. Journal of immunology (Baltimore, Md. : 1950), 195(4), 1399-1407.

+ Open protocol

+ Expand

Nardosinone Modulates Osteoclastogenesis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Nardosinone (Figure 1A), purchased from Must Biotechnology (Chengdu, China), was dissolved in dimethyl sulfoxide (DMSO). Alpha modification of Eagle’s minimum essential medium (α-MEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco BRL (Gaithersburg, MD, United States). Recombinant murine M-CSF and RANKL were purchased from R&D Systems (Minneapolis, MN, United States). Tartrate-resistant acid phosphatase (TRAP) staining solution, Triton X-100, and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) were obtained from Sigma-Aldrich (St. Louis, MO, United States). FITC phalloidin was obtained from Yeasen biotech Co., Ltd (Chengdu, China). Primary antibodies targeting GAPDH, IκBα, phospho-Akt, Akt, phospho-ERK1/2, ERK1/2, phospho-JNK1/2, JNK1/2, phospho-p38, p38, phosphor-PLCγ2, PLCγ2, c-Fos and NFATc1 were purchased from Cell Signaling Technology (Danvers, MA, United States). Dichlorofluorescin diacetate (DCFDA) cellular ROS detection assay kits were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). LPS from P. gingivalis was purchased from InvivoGen (San Diego, CA, United States).

Niu C., Xiao F., Yuan K., Hu X., Lin W., Ma R., Zhang X, & Huang Z. (2017). Nardosinone Suppresses RANKL-Induced Osteoclastogenesis and Attenuates Lipopolysaccharide-Induced Alveolar Bone Resorption. Frontiers in Pharmacology, 8, 626.

+ Open protocol

+ Expand

Western Blot Analysis of Cellular Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

The total protein in cells was isolated at various times. Then, the protein concentration of the lysates was measured using a BCA protein assay kit (Pierce, IL, USA). After heating in boiled water for 10 min in sample buffer (Bio-Rad, USA), equal aliquots of protein (15 mg) were fractionated by 10% SDS–polyacrylamide gel electrophoresis gels (Invitrogen, CA, USA), transferred onto nitrocellulose membranes, and blocked for 1 hour in 1× TBS-T (tris-buffered saline with Tween 20) with 1% bovine serum albumin. After washing three times, the membranes were incubated with primary antibodies against ERK, p-ERK, p38, p-p38, Akt, p-Akt, RANKL, OPG, NFATc1, c-fos, and β-actin (Cell Signaling, MA, USA) overnight and then incubated with a horseradish peroxidase–conjugated secondary antibody (Santa Cruz, CA, USA) at 37°C for 1 hour. A ChemiDoc XRS+ image system with Image Lab software (Bio-Rad, USA) was used to visualize the protein bands, and the protein expression levels were normalized against the level of β-actin for each sample.

Yuan B., Wang L., Zhao R., Yang X., Yang X., Zhu X., Liu L., Zhang K., Song Y, & Zhang X. (2020). A biomimetically hierarchical polyetherketoneketone scaffold for osteoporotic bone repair. Science Advances, 6(50), eabc4704.

+ Open protocol

+ Expand

Osteoclast Differentiation Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

α‐Minimum essential medium (α‐MEM) and foetal bovine serum (FBS) were obtained from Gibco; Thermo Fisher Scientific, Inc. Soluble recombinant mouse macrophage‐colony stimulating factor (M‐CSF) and recombinant mouse RANKL were obtained from R&D Systems, Inc. Tartrate‐resistant acid phosphatase (TRAP) was obtained from Sigma‐Aldrich and Merck KGaA. High purity Ti particles (average diameter. 1‐5 µm) were obtained from Johnson Matthey (cat. no., 00681). The antibodies (GAPDH, NFkB, C‐FOS, NFATc1, p‐p38,) were purchased from Cell Signaling Technology, Inc. Enzyme‐linked immunosorbent assay (ELISA) kits (IL‐6, IL‐1β, TNF‐α, IL‐10, Arg‐1, iNOS) were purchased from R&D Systems, Inc Flow cytometry anti‐mouse CD16/32‐PE (cat. no.553145) and anti‐mouse CD206‐Alexa 647 (cat. no.565250) were purchased from BioLegend Inc The p38α/β MAPK inhibitor (SB202190) was purchased from Selleck.

Ge Y., Feng K., Liu X., Zhu Z., Chen H., Chang Y., Sun Z., Wang H., Zhang J., Yu D, & Mao Y. (2020). Quercetin inhibits macrophage polarization through the p‐38α/β signalling pathway and regulates OPG/RANKL balance in a mouse skull model. Journal of Cellular and Molecular Medicine, 24(5), 3203-3216.

+ Open protocol

+ Expand

Western Blot Analysis of NFAT Proteins

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Western blotting was performed as described previously [67 (link)]. We used the following primary antibodies for our study: NFATc1 (8032S; Cell Signaling Tech), NFATc2 (sc-7296; Santa Cruz Biotech), NFATc3 (sc-8405; Santa Cruz Biotech), NFATc4 (sc-13036; Santa Cruz Biotech), C-MYC (sc-40; Santa Cruz Biotech), α-tubulin (T9026; Sigma), GAPDH (FL-335; Santa Cruz Biotech), and DDK (TA50011-100; OriGene).

Lee S.H., Kieu C., Martin C.E., Han J., Chen W., Kim J.S., Kang M.K., Kim R.H., Park N.H., Kim Y, & Shin K.H. (2019). NFATc3 plays an oncogenic role in oral/oropharyngeal squamous cell carcinomas by promoting cancer stemness via expression of OCT4. Oncotarget, 10(23), 2306-2319.

+ Open protocol

+ Expand

Western Blotting of Cell Signaling Proteins

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Standard protocols were followed for Western blotting assays (24 (link), 27 ). The primary antibodies used were pAkt, Akt, p GSK3β, GSK3β, NFATc1, cyclin D1 (Cell Signaling Technology, Danvers, MA, USA) and EF1α (Abcam, Cambridge, United Kingdom). Appropriate secondary antibodies were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA).

Pradhan A.K., Bhoopathi P., Talukdar S., Shen X.N., Emdad L., Das S.K., Sarkar D, & Fisher P.B. (2018). Recombinant MDA-7/IL-24 suppresses prostate cancer bone metastasis through down regulation of the Akt/Mcl-1 pathway. Molecular cancer therapeutics, 17(9), 1951-1960.

+ Open protocol

+ Expand

Quantitative PCR and Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

An RNeasy Plus kit (Qiagen, Germantown, MD, USA), a high capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA), and a Power SYBR Green PCR master mix kit (Applied Biosystems) were employed with listed PCR primers (Table 1). For detecting GFP-labeled 4T1.2 cells in the right femur, total DNA was isolated with QIAamp DNA mini kit (Qiagen) for qPCR.
For Western blotting, cells were lysed by a radio-immunoprecipitation assay buffer (Santa Cruz). Proteins were fractionated by 10-15% SDS gels and electro-transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Antibodies against ATF4, caspase 3, cathepsin K, eIF2α, p-eIF2α (Ser51), LC3A/B II, NFATc1, Chk1, p-Chk1 (Ser296) (Cell Signaling, Danvers, MA, USA), TRAP (Abcam, Cambridge, MA, USA), and β-actin (Sigma) were utilized.

Liu S., Liu Y., Minami K., Chen A., Wan Q., Yin Y., Gan L., Xu A., Matsuura N., Koizumi M., Liu Y., Na S., Li J., Nakshatri H., Li B.Y, & Yokota H. (2018). Inhibiting checkpoint kinase 1 protects bone from bone resorption by mammary tumor in a mouse model. Oncotarget, 9(10), 9364-9378.

+ Open protocol

+ Expand

Osteoclastogenesis Regulation by Signaling Pathways

Check if the same lab product or an alternative is used in the 5 most similar protocols

The alpha modification of Eagle’s medium (α-MEM), penicillin/streptomycin, and foetal bovine serum (FBS) were purchased from Gibco-BRL (Gaithersburg, MD, USA). The cell counting kit (CCK-8) was obtained from Dojindo Molecular Technology (Kumamoto, Japan). Recombinant mouse M-CSF and mouse RANKL were obtained from R&D (Minneapolis, MN, USA), and LY was purchased from Sigma Aldrich (St Louis, MO, USA). Specific antibodies against ERK, JNK, P38, IκB-α, phospho-ERK (Thr202/Tyr204), phospho-JNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyr182), MEK1/2, MKK6, MKK7, TAK1, phospho-MEK1/2, phospho-MKK6, phospho-MKK7, phospho-TAK1, NFATc1, and β-actin were obtained from Cell Signalling Technology (Cambridge, MA, USA). The TRAP staining kit, and all other reagents, were purchased from Sigma Aldrich unless otherwise stated.

Chen S., Jin G., Huang K.M., Ma J.J., Wang Q., Ma Y., Tang X.Z., Zhou Z.J., Hu Z.J., Wang J.Y., Qin A, & Fan S.W. (2015). Lycorine suppresses RANKL-induced osteoclastogenesis in vitro and prevents ovariectomy-induced osteoporosis and titanium particle-induced osteolysis in vivo. Scientific Reports, 5, 12853.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!