Total cell lysate was prepared in RIPA (Boston Bioproducts) containing protease and phosphatase inhibitor cocktail (Thermo). The cell lysate was quantified and proteins separated on SDS-PAGE and transferred to the PVDF (Bio-Rad) membrane. Membranes were blocked in 5% nonfat milk in TBS with 0.1% Tween-20 (TBST) for 1 h at room temperature followed by incubation with primary antibodies in TBST with 1% BSA overnight. The following primary antibodies were used – p Smad-2, p Smad-3, Smad-2, Smad-3, Snail, Vimentin, EPCAM, c-Myc, NFAT-1, Cyclin B, Cyclin D1, (Cell signaling Technology), Fibronectin, N-Cad (BD Biosciences), Pancytokeratin, GAPDH, α tubulin and β actin (Sigma), Acidic and basic Cytokeratin AE1/AE3 (Millipore), EMA (Dako), p27 (Santa Cruz Biotechnology), and cytokeratin-8 (Developmental Studies Hybridoma Bank, University of Iowa). Secondary antibodies (from sigma) were used at a concentration of 1:10,000. Equal loading was verified by immunoblotting with GAPDH, αTubulin or β Actin [44 (link)].
Nfat1
Manufactured by Cell Signaling Technology
Sourced in United Kingdom
NFAT1 is a transcription factor that plays a crucial role in the regulation of gene expression in various cell types. It is involved in the activation and proliferation of T cells, as well as the development and function of the immune system. NFAT1 is a key component of the calcium-calcineurin signaling pathway, which is essential for the activation of immune cells in response to antigen stimulation.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Lamp1, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Pan akt, by Cell Signaling Technology (2 mentions)
Sqstm1 p62, by Cell Signaling Technology (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Epcam, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Radioimmunoprecipitation assay (ripa), by Boston BioProducts (1 mentions)
Cyclin b, by Cell Signaling Technology (1 mentions)
N cad, by BD (1 mentions)
Pan cytokeratin, by Merck Group (1 mentions)
α tubulin and β actin, by Merck Group (1 mentions)
Acidic and basic cytokeratin ae1 ae3, by Merck Group (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
Gapdh, by Merck Group (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
15 protocols using nfat1
1
Western Blot Analysis of EMT Markers
2
Western Blot Analysis of Key Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, and supplemented with protease and phosphatase inhibitors) and shaken vigorously every 10 min for 3 h. Lysates were centrifugated (13000 rpm for 5 min), and supernatants were stored at −80°C until use. Proteins were quantified by Bradford protein assay and separated by SDS-PolyAcrylamide Gel Electrophoresis (SDS-PAGE). The proteins were transferred to nitrocellulose membranes (Bio-Rad), and membranes were blocked with 3% nonfat dry milk in TBS for 1 h. Primary antibodies incubated overnight at 4°C with the indicated primary antibody (Non-phospho (Active) β-catenin (Cat. No. 8814 Cell Signaling), NFAT1 (Cat. No. 610703 BD Biosciences), NFAT2 (Cat. No. 556602 BD Biosciences), NFAT3 (sc-271597 Santa Cruz Biotechnology), NFAT4 (SC-8405 Santa Cruz Biotechnology), Lamin B1 (ab16048 Abcam), α-Tubulin (T9026 Sigma)] according to manufacturer’s instructions. Then, membranes were washed with washing buffer (TBS, 0.025% Tween 20) and incubated with HRP-conjugated secondary antibody for 2 h at room temperature. Blots were revealed using SuperSignal Kit (Pierce) in C-DiGit Blot scanner (LI-COR Bioscience, Lincoln, NE, USA), and analysis was performed by Image Studio™ Lite Software (LI-COR Biosciences).
3
Comprehensive Immunoblot Antibody Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used for immunoblot: GAPDH (cat. 5174, 1:1,000), SQSTM1/p62 (cat. 5114, 1:1,000), TFEB (cat. 4240, 1:1,000), LAMIN A/C (cat. 2023, 1:1000), AMPKα-pThr172 (cat. 2535, 1:1,000), total AMPKα (cat. 5831, 1:1,000), ACC-pS79 (cat. 1:1000), total ACC (cat. 3676, 1:1,000), S6-pS235/236 (cat. 4858, 1:1,000), total S6 (cat. 2217, 1:1,000), Na+, K+ -ATPase α1 (cat. 3010, 1:1,000), p70-S6K-pThr389 (cat. 9234, 1:1,000), total p70-S6K (cat. 2708, 1:1,000), TSC2-pThr1462 (cat. 3611, 1:1,000), total TSC2 (cat. 3635, 1:1,000), AKT-pThr308 (cat. 2965, 1:1,000), pan AKT (cat. 4691, 1:1,000), IP3R1 (cat. 3763, 1:1,000), and TFE3 (cat. 14779, 1:1000) were from Cell Signaling Technology and PPP3CB (cat. ab191374, 1:1000) was from Abcam; the following antibody was used for immunofluorescence: TFEB (cat. sc-48784, 1:100) from Santa Cruz Biotechnology and NFAT1 (cat. 5861, 1:100) and LAMP1 (cat. 9091, 1:200) from Cell Signaling Technology; the following antibody was used for immunohistochemistry: p62/SQSTM1 (cat. ab91526, 1:200) from Abcam. Cells were collected in 2× sample buffer, boiled, and sonicated for immunoblot analysis using standard western blot protocols. Uncropped blots were shown in Supplementary Fig. 7 .
4
OVA-Induced BMDC Activation and NF-κB Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMDCs were seeded onto a 100 mm Petri
dish at a density of 1 × 106 cells per well and incubated
overnight. The cells were then treated with OVA (10 μg/mL) or
OVA (10 μg/mL) plus AnCHNPs (5 μg/mL). After incubation
for 24 h, cells were harvested and lysed with a RIPA buffer supplemented
with 1× proteinase inhibitor cocktail (Amresco). Protein concentration
was determined using a bicinchoninic acid (BCA) protein assay (Thermo
Fisher Scientific). Protein lysates were loaded onto 10% SDS-PAGE
and transferred to a PVDF membrane. Nonspecific binding to the membrane
was blocked by incubation with 5% nonfat milk at room temperature
for 1 h. The membrane was incubated with primary antibodies at 4 °C
overnight at dilutions specified by the manufacturer. This is followed
by incubation with secondary antibodies for 1 h at room temperature
and then treatment with ECL reagents (Thermo Fisher Scientific). The
membrane was then exposed to X-ray films (Santa Cruz). All the imaging
results were analyzed by ImageJ. The antibodies used are NFAT1 (Cell
Signaling Cat # 4389S); phospho-IκBα, phospho-NF-κB
p65 (Cell Signaling Cat # 9936T); and GAPDH (Cell Signaling Cat #
5174S).
dish at a density of 1 × 106 cells per well and incubated
overnight. The cells were then treated with OVA (10 μg/mL) or
OVA (10 μg/mL) plus AnCHNPs (5 μg/mL). After incubation
for 24 h, cells were harvested and lysed with a RIPA buffer supplemented
with 1× proteinase inhibitor cocktail (Amresco). Protein concentration
was determined using a bicinchoninic acid (BCA) protein assay (Thermo
Fisher Scientific). Protein lysates were loaded onto 10% SDS-PAGE
and transferred to a PVDF membrane. Nonspecific binding to the membrane
was blocked by incubation with 5% nonfat milk at room temperature
for 1 h. The membrane was incubated with primary antibodies at 4 °C
overnight at dilutions specified by the manufacturer. This is followed
by incubation with secondary antibodies for 1 h at room temperature
and then treatment with ECL reagents (Thermo Fisher Scientific). The
membrane was then exposed to X-ray films (Santa Cruz). All the imaging
results were analyzed by ImageJ. The antibodies used are NFAT1 (Cell
Signaling Cat # 4389S); phospho-IκBα, phospho-NF-κB
p65 (Cell Signaling Cat # 9936T); and GAPDH (Cell Signaling Cat #
5174S).
5
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted in a lysis buffer (Beyotime, Shanghai, China) and the nuclear proteins were extracted using a nuclear protein extraction kit (Beyotime). The denatured proteins were separated by 10% SDS-PAGE and then transferred to a PVDF membrane (Millipore, Billerica, MA, USA) by using a wet transfer apparatus (Bio-Rad). The membrane was then blocked in 5% (w/v) skim milk in phosphate buffered saline containing 0.1% Tween 20 (PBS-T) for 2 h at room temperature. Subsequently, the membrane was incubated with antibodies against ASIC2 (Abcam, Cambridge, UK), NFAT1 (Cell Signaling, Beverly, MA, USA), β-actin (Sigma, St Louis, MO, USA), or Histone H3 (Abcam) overnight at 4 °C. The HRP-coupled anti-rabbit secondary antibody was used at a final dilution of 1:5000 visualized with an enhanced chemiluminescence (ECL) detection system (Thermo Scientific, Waltham, MA, USA).
6
Western Blot Analysis of Bim and P-65
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the detection of Bim and P-65 protein, nuclear or cytoplasmic proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer's instructions. Samples were heated in sodium dodecyl sulfate sample buffer at 95 °C for 10 min, separated on a polyacrylamide gel, and separated proteins were electroblotted onto polyvinylidene difluoride membranes. Specific proteins were revealed by mouse and rabbit antibodies against NFAT1, p53, laminB or GAPDH (Cell Signaling Technology, Danvers, MA, USA) by overnight incubation at 4 °C, followed by chemiluminescent detection according to the manufacturer's instructions.
7
Subcellular Fractionation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with RIPA buffer. Nuclear and cytoplasmic fractions were isolated using NE-PER Nuclear and Cytoplasmic extraction reagents (Pierce). Protein concentrations were determined using the DCTM Protein Assay (Bio-Rad). Following SDS-PAGE, Western Blot analysis was performed using the following primary antibodies: β-catenin (Millipore), NFAT1 (Cell Signaling), pan-Akt (Cell Signaling), phospho-Akt (Ser473, Cell Signaling), TATA-binding protein (TBP, Abcam), and α-tubulin (Developmental Studies Hybridoma Bank). A horseradish peroxidase-conjugated antibody (Millipore) was used as a secondary antibody, and Pierece ECL Western Detection kit (ThermoFisher) was used for protein visualization.
8
Evaluating NFAT1 and CD8+ T-cells in Lung Tissue
9
Antibody Panel for Autophagy Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used in this study were as follows: ATG16L1 (8089, human), phospho-ATG14 S29 (92340), ATG14 (96752), phospho-Beclin S30 (54101), FIP200 (12436), FLCN (3697), GABARAPL1 (26632), GABARAPL2 (14256), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [5174, 1:10,000 for WB (Western blot)], DYKDDDDK tag (14793), HA tag (3724), myc tag (2278), LC3A/B (12741), LC3B (3868), LAMTOR1 (8975), LAMP1 [15665, 1:1000 for immunofluorescence (IF)], NFAT1 (5861, 1:250 for IF), NPRL2 (37344), phospho-S6K (9234), S6K (2708), phospho-S6 S235/236 (4858, 1:3000 for WB), S6 (2217, 1:5000 for WB), TAX1BP1 (5105), TFEB (4240), TFEB (37785, 1:200 for IF), and phospho-ULK S757 (14202) were from Cell Signaling Technology. Mouse monoclonal anti–S. Typhimurium lipopolysaccharide (clone 1E6, ab8274) and FNIP1 (ab134969) were from Abcam. TFE3 (HPA023881) was from MilliporeSigma. p62 (GP62-C) was from Progen. Galectin-3 (sc-23938) was from Santa Cruz Biotechnology. TFEB (A303-673A, 1:200 for IF in murine cells) was from Bethyl Laboratories. All antibodies were used at a 1:1000 dilution for Western blotting unless otherwise noted.
10
Western Blot Analysis of Chondrocyte Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates from the micromass cultures or primary chondrocytes that were treated with Ca-Pi were separated by SDS-PAGE and transferred onto PVDF membranes (Amersham Biosciences, Buckinghamshire, UK). The membranes were blocked with 5% skim milk at room temperature (RT) for 1 h, followed by incubation with the primary antibodies listed below for 1 h with shaking. After washing the membrane with TBS-T, the membranes were incubated with an HRP-conjugated secondary antibody (Santa Cruz Biotechnology, CA, USA) for 1 h and washed again. The signal on the membranes was visualized using enhanced chemiluminescence solution (Amersham Biosciences). The primary antibodies against Col2, Col10, Mmp-3, Mmp-13, Adamts5 and Runx2 were purchased from Abcam (Cambridge, UK); the phospho-Erk1/2, phospho-P38, phospho-JNK, phospho-Akt, phospho-P65, phospho-IkB, NFAT1 and NFAT3 antibodies were purchased from Cell Signaling Technology (Danvers, MA); and the Epas1 and NFAT4 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
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?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!