Anti nfatc1

Manufactured by Thermo Fisher Scientific

Sourced in China

Anti-NFATc1 is a laboratory reagent used in research applications. It is a monoclonal antibody that specifically binds to the transcription factor NFATc1, which plays a role in the regulation of gene expression. This product can be used in various experimental techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to study the expression and activity of NFATc1 in biological samples.

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Lab products found in correlation

Anti lamin a c, by Cell Signaling Technology (2 mentions) Trans blot turbo blotting system, by Bio-Rad (2 mentions) Criterion gel, by Bio-Rad (2 mentions) Odyssey clx imaging system, by LI COR (2 mentions) Anti ets2, by Santa Cruz Biotechnology (2 mentions) Anti myc, by Cell Signaling Technology (2 mentions) Anti rcan1, by Merck Group (2 mentions) Anti mkp3, by Santa Cruz Biotechnology (2 mentions) Anti erk1 2, by Cell Signaling Technology (2 mentions) Anti gfp, by Thermo Fisher Scientific (2 mentions) Anti flag, by Merck Group (2 mentions) Anti gapdh, by Biosynth (2 mentions) A32961, by Thermo Fisher Scientific (1 mentions) Anti phospho erk1 2, by Cell Signaling Technology (1 mentions) Alexa fluor 594 secondary antibody, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Hoechst 33258, by Merck Group (1 mentions) Tcs sp8, by Leica (1 mentions) Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions) Anti p erk1 2, by Cell Signaling Technology (1 mentions)

4 protocols using anti nfatc1

NFATc1 Activation Assay in RAW 264.7 Cells

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To detect activation of NFATc1, RAW 264.7 cells were treated with the TRAIL (500 ng/ml), M-CSF (20 ng/ml), and RANKL (50 ng/ml) in the presence or absence of TRAIL-R siRNA or anti-TRAIL-R. After stimulation, cell lysates of the nuclear fraction were prepared. Nuclear and cytosolic proteins were separated upon resuspension of pelleted cells (10⁶) in 30 ml of hypotonic lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl₂, and 0.5% NP-40) by gentle pipetting, then incubation on ice for 5 min. The homogenate was centrifuged at 6000 rpm for 5 min at 4 °C. The supernatant representing the cytosolic fraction was collected and stored at –80 °C, and the pellet containing cell nuclei was lysed in 30 ml RIPA lysis buffer. The nuclear extract (supernatant) was retained after centrifugation at 13,000 × g for 5 min at 4 °C for subsequent western blotting with anti-NFATc1 (Thermo Scientific, Rockford, IL) and anti-histone deacetylase (HDAC) antibodies (Cell Signaling, Danvers, MA).

Liao H.J., Tsai H.F., Wu C.S., Chyuan I.T, & Hsu P.N. (2019). TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment. Cell Death & Disease, 10(2), 77.

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Protein Fractionation and Western Blot Analysis

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Proteins were prepared with radioimmunoprecipitation assay buffer (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 0.25% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors (A32961; Thermo Fisher). Nuclear and cytoplasmic fractions were isolated using a kit according to the manufacturer’s instructions (78835; Thermo Fisher). A total of 20 to 40 µg of proteins were separated using Criterion gels (Bio-Rad) and transferred to nitrocellulose membranes using the Trans-Blot Turbo Blotting System (Bio-Rad). The membranes were then blocked with 5% nonfat milk or bovine serum albumin, followed by incubation with primary antibodies overnight at 4°C. After 3 washes, the membranes were incubated with secondary antibody and scanned with an Odyssey CLx Imaging system (LI-COR Biosciences). The following antibodies were used: anti-ETS2 (sc-365666; Santa Cruz Biotechnology), anti-phospho-ETS2 (44-1105G; Thermo Fisher), anti-GAPDH (10R-G109a; Fitzgerald), anti-RCAN1.4 (D6694; Sigma), anti-MKP3 (sc-137246; Santa Cruz Biotechnology), anti-lamin A/C (2032; Cell Signaling), anti-NFATc1 (nuclear factor of activated T cells 1; MA3-024; Thermo Fisher), anti-GFP (green fluorescent protein; A-11120; Invitrogen), anti-FLAG (7425; Sigma), anti-Myc (2276; Cell Signaling), anti-Erk1/2 (9107; Cell Signaling), and anti-phospho-Erk1/2 (9101; Cell Signaling).

Luo Y., Jiang N., May H.I., Luo X., Ferdous A., Schiattarella G.G., Chen G., Li Q., Li C., Rothermel B.A., Jiang D., Lavandero S., Gillette T.G, & Hill J.A. (2021). Cooperative Binding of ETS2 and NFAT Links Erk1/2 and Calcineurin Signaling in the Pathogenesis of Cardiac Hypertrophy. Circulation, 144(1), 34-51.

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Immunofluorescence Imaging of NFAT Transcription Factors

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The method has been widely implemented by our laboratories [24 ]. The cells on coverslips were probed with Anti-NFATc1 (MA3–024, Thermofisher), Anti-NFATc2 (JA11–08, HuaAn Biotechnology Co., Ltd., Hangzhou, China), Anti-NFATc3 (sc-8405, Santa cruz), and Anti-NFATc4 (YT3085, Immunoway, Suzhou, China) primary antibody. After the incubation with an Alexa Fluor® 488 (for primary antibody of Anti-NFATc1 and anti-NFATc3) (Life Technologies, Massachusetts, USA) for and an Alexa Fluor® 594 secondary antibody (for primary antibody of anti-NFATc2 and anti-NFATc4) (Life Technologies, Massachusetts, USA), the cells were stained with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO, USA) for nucleus label. Finally, the cells were visualized with a confocal microscope (Leica Microsystems TCS SP8. Wetzlar, Germany). Control samples without adding the primary antibody were prepared for determining the level of non-specific noise.

Huang J., Hu M., Niu H., Wang J., Si Y., Cheng S, & Ding W. (2021). Osteopontin isoform c promotes the survival of cisplatin-treated NSCLC cells involving NFATc2-mediated suppression on calcium-induced ROS levels. BMC Cancer, 21, 750.

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Western Blot Analysis of Protein Signaling

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Proteins were prepared with radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl PH 7.4, 150 mM NaCl, 1mM EDTA, 0.25% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors (Thermo Fisher, A32961). Nuclear and cytoplasmic fractions were isolated using a kit according to the manufacturer’s instructions (Thermo Fisher, 78835). 20–40 μg of proteins were separated using Criterion gels (Biorad) and transferred to nitrocellulose membranes using the Trans-Blot^® Turbo^™ Blotting System (Biorad). The membranes were then blocked with 5% non-fat milk or BSA, followed by incubation with primary antibodies overnight at 4°C. After 3 washes, the membranes were incubated with secondary antibody, and scanned with an Odyssey CLx Imaging system (LI-COR). The following antibodies were used: anti-ETS2 (Santa Cruz Biotechnology, sc-365666), anti-p-ETS2 (Thermo Fisher, 44–1105G), anti-GAPDH (Fitzgerald, 10R-G109a), anti-Rcan1.4 (Sigma, D6694), anti-MKP3 (Santa Cruz Biotechnology, sc-137246), anti-Lamin A/C (Cell Signaling, 2032), anti-NFATc1(Thermo Fisher, MA3–024), anti-GFP (Invitrogen, A-11120), anti-flag (Sigma, 7425), anti-myc (Cell Signaling, 2276), anti-Erk1/2 (Cell Signaling, 9107), anti-p-Erk1/2 (Cell Signaling, 9101).

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