Transfected cells were collected and lysed with 2× Lysis Buffer (Sigma–Aldrich, St. Louis, Missouri, U.S.A.). Total proteins in the cell or tissue samples were quantitated using BCA protein assay kit (Pierce Biotechnology, Rockford, IL, U.S.A.) following the instructions. The obtained proteins were first separated using 10% SDS/PAGE and then transferred on to polyvinylidene fluoride membranes (PVDF, Invitrogen, Carlsbad, CA). The membranes were blocked using TBST solution which was supplemented with 5% skim milk at 4°C for 1 h, and then incubated with first antibodies against AKT3 (1:1000, CST#9272, Boston, U.S.A.), NFATc2 (1:500, Abcam ab2722, Cambridge, U.K.), PPP3CA (1:2000, Abcam ab3673, Cambridge, U.K.), FOS (1:200, Abcam ab7963, Cambridge, U.K.) and AKT1S1 (1:1000, CST#2691, Boston, U.S.A.) overnight at 4°C, followed by the incubation with second antibodies for 1.5 h at room temperature. The blots were visualized with enhanced chemiluminescence kit (GE Healthcare, Chalfont St. Giles, Buckinghamshire, U.K.) following the manufacturer’s protocols.
Ab2722
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab2722 is a lab equipment product. It is a general-purpose tool used in scientific research and laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Ab2796, by Abcam (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Ab83832, by Abcam (3 mentions)
Sab4501982, by Merck Group (3 mentions)
Ab6002, by Abcam (3 mentions)
Erα antibody, by Cell Signaling Technology (2 mentions)
Ab128008, by Abcam (2 mentions)
Sc 376157, by Santa Cruz Biotechnology (2 mentions)
Sc 20641, by Santa Cruz Biotechnology (2 mentions)
Enhanced chemiluminescence kit, by GE Healthcare (1 mentions)
Polyvinylidene fluoride (pvdf), by Thermo Fisher Scientific (1 mentions)
Ab3673, by Abcam (1 mentions)
Ab7963, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Gapdh antibody, by Cell Signaling Technology (1 mentions)
Ab38646, by Abcam (1 mentions)
Ab32518, by Abcam (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ab205718, by Abcam (1 mentions)
20 protocols using ab2722
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Key Proteins
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Cells were lysed using radioimmunoprecipitation (RIPA) lysis buffer (Beyotime, Shanghai, China). Protein concentrations were detected using bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). Antibodies used in the assays were NFAT1 antibody (ab2722, Abcam, Cambridge, UK), ER-α antibody (#8644, Cell Signaling Technology) and GAPDH antibody (#5174, Cell Signaling Technology), IRF9 antibody (#76684, Cell Signaling Technology), Methyl-CpG binding domain protein 2 (MBD2) antibody (ab38646, Abcam) and IL-17 antibody (ab77171, Abcam).
3
Western Blot Analysis of NFAT and NF-κB Signaling
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The brain tissues were added with pre-cooled Tris-HCl buffer (pH 7.4) at the ratio of 1:3, and made into brain tissue homogenate with a glass homogenizer in an ice bath, and then the cells or tissue homogenates were lysed in cold RIPA containing a protease inhibitor cocktail [50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS, 1 mM PMSF, 1 mM NaF, 1 mM Na3VO4, 1% protease inhibitor cocktail from Sigma, which was added right before use], for 30 min. The lysate was then centrifuged at 4°C for 20 min at 16,000 × g to collect the supernatant. The protein concentration was determined using the Pierce bicinchoninic acid assay kit (Beyotime Biotechnology Co., Ltd.). Equal amounts of protein (20 μ.l/well) were separated by 7.5% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk for 1 h and incubated overnight at 4°C with primary antibodies as follows: Recombinant nuclear factor of activated T cells c2 (NFATc2) (1:1,000, ab2722; Abcam), p65 (1:1,000, ab16502; Abcam), p-p65 (1:2,000, ab86299; Abcam), IκBα (1:1,000, ab32518; Abcam), and p-IκBα (1:10,000, ab133462; Abcam). The membranes were then incubated with secondary antibody IgG (1:2,000, ab205718; Abcam). The gray value of the target band was analyzed using ImageJ software (Rawak Software, Inc.) with β-actin as the internal reference.
4
Immunofluorescence Staining of CTSK and Nfatc1 in Mouse Maxillae
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After decalcification, the mouse maxillae were embedded in optimal cutting temperature compound (OCT; Tissue-Tek, USA) and sliced at a thickness of 8 μm using a CM1850 (Leica, USA). Then, the sections were incubated with hyaluronidase (HA; Sigma, USA) for 1 h at 37 °C, followed by incubation with goat serum (MXB, China) for 1 h. After incubation with anti-CTSK antibody (ab19027; dilution 1:400; Abcam, USA) overnight at 4 °C, the sections were incubated with Alexa Flour 586 IgG (ab 150088; dilution 1:1 000; Abcam, USA) for 1 h. Finally, the sections were counterstained with DAPI (Sigma, USA) for 10 min. For immunofluorescent cellular assays, before staining, the cells were fixed with 4% PFA (Aladdin, China) for 30 min and incubated with 0.1% Triton X-100 (BBI, China) for 20 min. Then, goat serum (MXB, China) was used to block the cells. Subsequently, the cells were co-incubated with anti-Nfatc1 antibody (ab2722; dilution 1:400; Abcam, USA) and anti-actin antibody (ab8227; dilution 1:500, Abcam, USA) overnight at 4 °C. Alexa Flour 488 IgG (ab150113; dilution 1:1 000; Abcam, USA) and Alexa Flour 586 IgG (ab 150088; dilution 1:1 000; Abcam, USA) were used to visualise Nfatc1 and actin in the cells. Finally, the nuclei were counterstained with DAPI (Sigma, USA). An inverted fluorescence microscope (Nikon, Japan) was used to obtain images.
5
ChIP assay for NFAT1 and ER-α
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ChIP assay was conducted using EZ ChIP kit (Millipore, Billerica, MA, USA) and NFAT1 antibody (ab2722, Abcam) or ER-α antibody (#8644, Cell Signaling Technology) according to the instruction of the manufacturer. The primers specific to HERV-E clone 4–1 5′ LTR were: 5′-CTCCCCAACCTCCCCTTTTC-3′ and 5′-TGAGAAACATGACTGGGGGC-3′. Normal rabbit IgG (A7016, Beyotime, Shanghai, China) was used to control the nonspecific immunoprecipitation.
6
NFAT1 ChIP-seq Protocol for BMI1 Promoter
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ChIP assay was performed as our previously described [27 (link),29 (link)]. Briefly, treated MCF-7 cells at 90% to 100% confluence in 10-cm dishes were fixed with 1% formaldehyde and sonicated into 100- to 500-bp fragments. Soluble chromatin was precipitated with anti-NFAT1 (Abcam: ab2722) or a control non-immune IgG (Cell Signaling Technology: #2729). For real-time ChIP-PCR, the SYBR green system was used with the ABI Prism 7300 sequence detector (Applied Biosystems). Data are reported as relative fold enrichment. Primers used for ChIP qPCR of the BMI1 promoter are in Table S1.
7
ChIP and Western Blot Antibody Panel
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The following antibodies were used for the ChIP experiment: rabbit polyclonal anti-PARP (ab6079, Abcam), anti-H2AX (#39689, Active motif), anti-H2A.Z (#39943, Active motif), mouse anti-NFATC2 (ab2722, Abcam) and anti-H2A (Active motif, #39111). For western blot analysis, anti-PARP-1 (C2–10, Trevigen), anti-H3 (Millipore), anti-H3S10 (Millipore), anti-H3T3 (Millipore), mouse monoclonal anti-α tubulin (Sigma), anti-NFATC2 (Thermo, #PA5-19164), anti-RNAPolII (Covance, 8WG16) and anti-pADPr (Trevigen, 10H) were used. For electron microscopy (EM) immunocytochemistry, anti-H1 antibody (sc8030, Santa Cruz) and anti-PARP-1 (ab6079, Abcam) were used. Either goat anti-rabbit or anti-mouse secondary antibodies conjugated to horseradish peroxidase (Sigma) were used.
8
Neonatal Cardiomyocyte Protein Interaction
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Protein extracts of neonatal rat cardiomyocytes were prepared with RIPA buffer. Protein extracts (1 mg) were immunoprecipitated overnight at 4°C with 5 μg of anti-NFAT (ab 2722, Abcam, Cambridge, UK) or 5 μg of anti-MEF-2c (C-21X sc313x, Santa Cruz Biotechnology Inc., CA, USA) antibodies. Immunoprecipitated proteins were recovered using Protein A Sepharose CL-4B (17-0780-01, GE Health Care, IL, USA). Immunoprecipitated proteins were analyzed by Western blot with anti-MEF-2c (C-21X sc313x, Santa Cruz Biotechnology Inc., CA, USA) antibody.
9
Western Blot Analysis of Protein Translocation
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Total protein was extracted from cells using lysis buffer. To assess NFAT1 nuclear translocation, cytoplasmic and nuclear fractions were prepared as previously described (Diaz-Meco et al, 1999 (link); Gomez-Sintes and Lucas, 2010 (link)). Equal amounts of protein from each sample were separated by electrophoresis and transferred to a nitrocellulose membrane, which was blocked and incubated overnight at 4 °C with antibodies against GSK-3α/β (D75D3; Cell Signaling Technology, Beverly, MA, USA), pSer21/9-GSK-3α/β (D17D2; Cell Signaling Technology), p53 (ab1101; Abcam, Cambridge, UK), Fas (ab103551; Abcam), NFAT1 (ab2722; Abcam) and FasL (all at 1 : 1000 dilution). The membrane was then incubated with the appropriate secondary antibody. Protein bands were detected using an enhanced chemiluminescence kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and band intensity was quantified using Sigma-Gel software (Jandel Scientific Software, Sari Kafael, CA, USA).
10
Chromatin Immunoprecipitation Assay of Spinal Cord
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ChIP assay was performed using the ChIP Assay Kit (CST, 9005). The animal’s L4 and L5 spinal cords were removed quickly and placed in 1% formaldehyde for 10 min at room temperature to cross-link transcription factors with chromatin. 125 mM glycine was then added to inactivate the formaldehyde. After adding micrococcal nuclease to digest DNA to the length of approximately 150–900 bp, DNA fragments were immunoprecipitated using antibodies targeting NFATc2 (5 μg, Abcam, ab2722), ac-H3 (5 μg, Abcam, ab32129), ac-H4 (5 μg, Sigma, 06-598), H4K20me3 (5 μg, Epigentek, A-4048) or IgG overnight at 4°C. The next day, ChIP-grade protein G magnetic beads were added and incubated for 2 h at 4°C with rotation. Then the chromatin-protein-antibody-bead complexes were eluted, and the DNA was extracted. The precipitated DNA was resuspended in the nuclear-free water, and qPCR assays with primers were performed to amplify the different regions within the TSC2 promoter. Finally, the ratio of ChIP/input in the spinal dorsal horn was calculated.
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