CD4+ T cells were lysated and proteins were extracted using a nuclear extraction reagent (Boster Biological Technology, Pleasanton, CA, USA). Proteins were quantified by the Bradford reagent (Thermo Fisher Scientific, Waltham, MA, USA), followed by 12% vertical dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred into a polyvinylidene difluoride (PVDF) membrane (Sigma-Aldrich, St. Louis, MO, USA). The PVDF membrane was blocked in 5% skim milk for 1 h at room temperature, then incubated with an antibody against P65 (GB11142, 1:1000, Wuhan Servicebio Technology Co., Ltd., Wuhan, China) or P50 (ab7971, 1:5000, Abcam, Cambridge, MA, USA) for 12-16 h at 4℃ , and followed by incubating with a mouse anti-rabbit IgG antibody (H&L) (GenScript, Piscataway, NJ, USA). Proteins were detected with an enhanced chemiluminescence (ECL) western blot detection kit (Thermo Fisher Scientific, Waltham, MA, USA). Quantification of P65 and P50 was normalized to GAPDH by densitometry.
Ab7971
Manufactured by Abcam
Sourced in United States
Ab7971 is a primary antibody targeting the Synaptotagmin-1 protein. Synaptotagmin-1 is a calcium-sensing protein that regulates synaptic vesicle exocytosis and neurotransmitter release.
Automatically generated - may contain errors
Lab products found in correlation
Ab7970, by Abcam (3 mentions)
Ab10016, by Sangon (2 mentions)
Bs 1165r, by Bioss Antibodies (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bradford reagent, by Thermo Fisher Scientific (1 mentions)
Gb11142, by Wuhan Servicebio Technology (1 mentions)
Nuclear extraction reagent, by Boster Bio (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Image lab 6, by Bio-Rad (1 mentions)
Sc 55529, by Santa Cruz Biotechnology (1 mentions)
M iggκ bp, by Santa Cruz Biotechnology (1 mentions)
Sc 7271, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Sc 516102, by Santa Cruz Biotechnology (1 mentions)
Sc 2004, by Santa Cruz Biotechnology (1 mentions)
Anti nicd 4147, by Cell Signaling Technology (1 mentions)
Ab5131, by Abcam (1 mentions)
Ap1022a, by Abcepta (1 mentions)
Sc 2027, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
14 protocols using ab7971
1
Nuclear Extraction and Western Blot Analysis
2
Measuring NF-κB and iNOS Protein Levels
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The expression levels of transcription factor NF-κΒ and iNOS protein in the liver homogenate were measured by immunoblotting. Briefly, 50 µg of protein was resolved on 10% SDS polyacrylamide gels, and then transferred to polyvinylidene fluoride membranes and blocked with 5% (w/v) nonfat milk in TBS-T (10 mM Tris/HCl, 150 mM NaCl (pH 7.6) and 0.05% TWEEN 20) for 1 h at 20 °C. Next, the membranes were incubated overnight at 4 °C on a shaker with primary anti- NF-κΒ (1:400; ab-7971, Abcam, Cambridge, UK), anti-NOS2 (1:200; sc-7271, Santa Cruz, CA, USA), or anti-β-tubulin (1:200; sc-55529, Santa Cruz, CA, USA). The samples were incubated for 2 h or overnight at 4 °C on a shaker with HRP-conjugated goat anti-rabbit IgG (1:4000; sc-2004, Santa Cruz, CA, USA) or m-IgGκ BP (1:1000; sc-516102, Santa Cruz, CA, USA). Images were acquired using chemiluminescence and a ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA), and were analyzed with ImageLab 6.0.1. (Bio-Rad, Hercules, CA, USA). The relative protein levels were calculated based on β-tubulin expression as a loading control.
3
Chromatin Immunoprecipitation and Real-time PCR
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ChIP was performed as described previously89 (link). Anti-JMJD3 antibody (AP1022a, Abgent), anti-NICD (4147, Cell Signaling), anti-trimethyl H3K27 antibody (07–449, Millipore), anti-monomethyl H3K27 antibody (07–448, Millipore), anti-phosphorylated RNA polymerase II antibody (ab5131, Abcam), anti-p50 antibody (ab7971, Abcam), anti-p65 antibody (ab7970, Abcam), or normal IgG (SC-2027, Santa Cruz Biotechnology) were used. Real-time PCR was performed with a Stratagene Mx3000P using primers (see Supplementary Table S2 ).
4
Quantitative Immunoblotting of PPAR-γ and P50
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At least 3 × 106 monocytes were lysed in radio immunoprecipitation assay (RIPA) buffer containing a proteinase and phosphatase inhibitor (Beyotime) for 30 min at 4°C. Lysates were centrifuged for 15 min at 14,000g at 4°C and the supernatant was discarded. Protein concentration in cell lysates were measured via the Bradford assay (HyClone-Pierce, USA). Proteins were separated by electrophoresis using 12% vertical dodecyl sulfate-polyacrylamide gel and transferred onto nitrocellulose membranes (Millipore, USA). The PVDF membranes was immersed in 5% skim milk for 1 h at room temperature and then immunoblotted with primary antibodies, icluding rabbit anti-human P50 (ab7971, 1:5,000, Abcam, USA) or mouse anti-human PPAR-γ antibody (ab41928, 1:1,000, Abcam, USA) for 12–16 h at 4°C, followed by incubation with the secondary Goat anti-mouse or anti-rabbit IgG antibody (H&L) (GenScript, USA). The band intensity was measured by an ECL Western blot detection kit (Thermo Scientific, USA). The expression level of PPAR-γ and P50 was quantified by densitometry with normalization to GAPDH.
5
Quantifying Cell Proliferation and Apoptosis
6
Pulldown Assay for NF-κB Subunit p50
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For one pull-down reaction, 1 × 106 T cells polarized and expanded as described above and nuclear extracts were generated as described in the section ‘Electrophoretic mobility shift assays', using 1 ml Hypotonic Buffer and 90 μl Nuclear Buffer for 1 × 106 T cells. Binding reactions of nuclear extracts to DNA probes were performed as in EMSA reactions (using the Thermo Scientific buffers) scaled up 20 times for one pull-down reaction, using 90 μl nuclear extracts of T cells plus 12 μl Bcl-3-overexpressing 293 T lysates or empty 293 T lysates. DNA probes were the same sequences as used in EMSAs, biotin-labelled. Binding reactions were incubated for in total 1 h, then 40 μl Streptavidin beads were added (Invitrogen, catalogue number: 65001) and rotated overnight at 4 °C. Proteins were eluted the next day with 2 × SDS Lämmli Buffer and visualized via western blotting with anti-p50 antibodies (Abcam ab7971). DNA sequences used in EMSAs and pulldowns are described in Table 2 .
7
Western Blot Analysis of Apoptotic Markers
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SW480 cells were collected at the treatment time-points with reagents as suggested in each experiment. Cells were lysed with RIPA buffer (Thermo Fisher Scientific) and 15% electrophoresis (Thermo Fisher Scientific) was conducted under 90 V for 2 h. After undergoing electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes. Primary antibodies, rabbit polyclonal anti-fibronectin antibody (1:500), rabbit polyclonal caspase-3 antibody (1:500, ab2302), rabbit polyclonal NF-κB antibody (1:500, ab7971), rabbit polyclonal p53 antibody (1:500, ab1431), rabbit polyclonal PARP (1:500, ab6079), rabbit polyclonal Bax antibody (1:500, ab53154), rabbit polyclonal cytochrome c antibody (1:500, ab90529) and rabbit polyclonal GAPDH antibody (1:500, ab9485) (all from Abcam) were incubated with the membranes for 24 h at 4°C. After washing with TBST buffer three times, the secondary antibody goat anti-rabbit IgG H&L (HRP) was incubated at room temperature for 1 h. Immunostaining was carried out using DAB Plus substrate and chemiluminescence system (Amersham Biosciences, Freiburg, Germany). The results were analyzed using chemiluminescence Molecular Imager® ChemiDoc™ XRS+ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
8
Western Blot Analysis of NF-kB Pathway
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Expression of NF-kB p50, P65, MARCH7, E-cadherin and beta catenin protein was analyzed by the Western blot method [9 ]. The primary antibodies used included polyclonal rabbit anti-MARCH7 (1:1000; ab84130; Abcam Inc., Cambridge, MA, USA); polyclonal rabbit anti-NF-kB p65 (1:1000; ab7970; Abcam Inc., Cambridge, MA, USA); polyclonal rabbit anti-NF-kB p50 (1:1000; ab7971; Abcam Inc., Cambridge, MA, USA); anti-beta catenin antibody rabbit polyclonal antibody (1:500, bs-1165R, Bioss, Beijing, China) and polyclonal rabbit anti-GAPDH (1:1000; AB10016; Sangon Biotech, Shanghai, China). The band density was analyzed using a gel imaging system and compared against an internal control.
9
NF-κB Signaling Pathway Profiling
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Mouse gastrocnemius muscle was washed twice with ice cold PBS and homogenized in 500 μl RIPA lysis buffer followed by three rounds of freeze-thaw. Tissue lysates were centrifuged for 5 min at 14,000 rpm at 4°C, and cleared supernatant was transferred into a new tube. For immunoprecipitations, 750 μg total protein was incubated with 5 μg anti NF-κB p65 (ab16502, Abcam), p50 (ab7971, Abcam) antibodies, or normal rabbit IgG (negative control), respectively, at 4°C overnight with rotation. Immune complexes were captured with 25 μl of protein A/G resin (Pierce) and incubated for 1 h at 4°C with rotation. Samples were washed four times with lysis buffer with centrifugation for 5 min at 2000 rpm at 4°C. After final wash, the pellets were boiled 5 min in 40 μl of 2× SDS sample buffer and subjected to SDS-PAGE and Western blot analysis.
10
Quantitative Protein Analysis by Western Blot
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Total protein was extracted using cell lysis buffer, and the protein concentration was determined using the BCA assay (both from Beyotime). Protein (100 µg) was subjected to SDS-PAGE, and then transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked for 2 h in 5% skimmed milk (Difco Laboratories, Detroit, MI, USA). Then, a membrane was incubated with the primary antibodies, including polyclonal rabbit anti-MARCH1 antibody (1:200; bs-9335R; Bioss), polyclonal rabbit anti-NF-κB p65 (1:1,000; ab7970), polyclonal rabbit anti-NF-κB p50 (1:1,000; ab7971) (both from Abcam Inc., Cambridge, MA, USA), polyclonal rabbit anti-β-catenin antibody (1:500; bs-1165R; Bioss), polyclonal rabbit anti-Histone H1, and polyclonal rabbit anti-E-cadherin antibody, overnight at 4°C. The membrane was incubated with the HRP-conjugated secondary antibody for 2 h. GAPDH was detected with a polyclonal antibody and served as the reference (1:1,000; AB10016; Sangon Biotech, Shanghai, China). Proteins were visualized with the ECL system (Beyotime) using the ChemiDoc XRS system (Bio-Rad).
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