HL‐60 and NB4 cells (~3 × 106) were washed, detached and suspended in cell lysis buffer for 30 min on ice. Cell lysates were collected, and 10% of the cell lysates were used as the input. The remaining lysates were aliquoted into two parts for IP with rabbit anti‐human Bmal1 (3 μg, Abcam) and an isotype control IgG (3 μg, Abcam). Samples were incubated at 4°C overnight, and Protein A/G Magnetic Beads were added and incubated for 2 h. Magnetic beads were washed, and immunoprecipitated complexes were recovered and subjected to western blotting to detect Bmal1 (1:1000, Abcam) and EZH2 (1:1000, Abcam).
Bmal1
Manufactured by Abcam
Sourced in United States, United Kingdom
BMAL1 is a protein that is a core component of the circadian clock, which is the internal biological clock that regulates various physiological processes in organisms. BMAL1 plays a crucial role in the regulation of circadian rhythms by forming a transcriptional-translational feedback loop with other clock proteins.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (2 mentions)
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (2 mentions)
Nf κb, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Chemiluminescence reagent, by Merck Group (1 mentions)
P nf κb, by Abcam (1 mentions)
Nf κb p65, by Abcam (1 mentions)
P mtorc1, by Abcam (1 mentions)
Mtorc1, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Biomax film, by Kodak (1 mentions)
Src 2, by Fortis Life Sciences (1 mentions)
Grp78, by Cell Signaling Technology (1 mentions)
Psmd9, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Calnexin, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Las 4000, by Fujifilm (1 mentions)
Antibodies against gapdh, by Abcam (1 mentions)
23 protocols using bmal1
1
Bmal1-EZH2 Coimmunoprecipitation Assay
2
Quantifying Protein Signaling Pathways
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After centrifugation (10,000 g , 15 min at 4°C), the supernatants were collected for protein analysis. The protein concentration was determined by Bradford protein assay. Western blot analysis was performed as previously described. Briefly, 50 µg proteins from each sample were loaded onto an SDS polyacrylamide gel for electrophoresis and subsequently transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat dry milk in PBS for an hour and incubated with a primary antibody against BMAL1 (1:200 dilution), AKT (1:1000 dilution), p-AKT (Thr 308) (1:2000 dilution), mTORC1 (1:1000 dilution), p-mTORC1 (Ser2481) (1:2000 dilution), S6K1 (1:1000 dilution), p70-S6K1 (Thr389) (1:2000 dilution), NfκBp65 (dilution 1:250 dilution), p-NfκB (Ser536) (1:250 dilution) overnight at 4°C (BMAL1, mTORC1, p-mTORC1, NfκBp65 and p-NfκB were purchased from Abcam; AKT, p-AKT, S6K1S and p70-S6K1 were purchase from CST Danvers, USA). Samples were then incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, USA) (1:5000 dilution). Band intensities were visualized with chemiluminescence reagent (Millipore Corp.) by the BioMax film (Kodak) and then analyzed with Gel-Pro analyzer 4.0 software. β-Actin (Santa Cruz, USA) protein was utilized as a loading control.
3
Temporal Chromatin Profiling in Liver
4
Protein Expression Analysis in Frozen Liver Tissue
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Frozen liver tissue was homogenized with in ice-cold RIPA buffer (50 mM Tris–HCl, pH7.4, 1% Nonidet P-40, 1% Sodium deoxycholate, 0.15 M NaCl) supplemented with protease and phosphatase inhibitors (Roche, Indianapolis, IN) on ice. The supernatant was collected after centrifugation at 4°C, 12,000×g for 10 minutes. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel followed by transfer to PVDF membranes. Target proteins were detected with the following specific monoclonal or polyclonal antibodies: Actin (MP Biomedicals, Solon, OH); Bmal1 (Abcam, Cambridge, MA); Psmd9 (Sigma, St. Louis, MO). Grp78, Pdi, Sirt3 and LC3 from Cell signaling (Danvers, MA). Actin was quantified as a loading control. Images were analyzed by Imagelab software (Bio-rad, Hercules, CA). Data were expressed as means ± standard error of the mean (SEM).
5
Autophagy Flux Quantification Protocol
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Proteins were quantified by immunoblotting using rabbit antibodies against LC3B, ULK1 (Cell Signaling Technologies, Danvers, MA, USA), PER2 (Millipore, Darmstadt, Germany) and BMAL1 (Abcam, Cambridge, UK). Gel loading was equalized according to protein content of the samples and controlled by amido black staining of the blotted membranes or parallel immune staining using rabbit and mouse beta-Actin or rabbit Calnexin antibodies (Sigma Aldrich, Martinsried, Germany). Western blots were developed with Peroxidase-coupled secondary antibodies. Chemo-luminescence was quantified with a digital camera system (LAS 4000, Fuji, Düsseldorf, Germany). Autophagy flux was determined according to current guidelines by treating cells with chloroquine (50 μM, 3 h) or bafilomycin (10 nM, 3 h) prior to the analysis of LC3-II protein levels. Autophagic flux values were calculated by subtracting LC3-II levels in untreated cells from LC3-II levels in cells treated with the lysosome inhibitors Chloroquine or Bafilomycin [16 (link)].
6
Aging and Senescence Pathway Analysis
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SBG was purchased from Biopurify Phytochemicals (Chengdu, China). D-galactose (D-gal) was purchased from Aladdin (Shanghai, China). Vitamin C was obtained from Yuanye Biotechnology (Shanghai, China). Biochemical kits for SOD, MDA, ROS and HYP were purchased from Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Staining kit for senescence-associated-β-galactosidase (SA-β-gal) was obtained from Beyotime Biotechnology (Shanghai, China). Antibodies against GAPDH, MMP-1, p53, BMAL1, REV-ERBɑ, BHMT and NLRP3 were purchased from Abcam (Cambridge, United Kingdom).
7
Western Blot Analysis of Cell Signaling
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Western blot analysis was performed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as previously described [21 (link)]. Antibodies were used against BMAL1 (Abcam, Cambridge, UK, ab3350), E-cadherin (Cell signaling Technology, Inc., Boston, MA, USA, 14472), vimentin (Cell signaling Technology, 73260), β-catenin (Cell signaling Technology, 8480). HSC-70 (Stressgen, CA, USA, SPA-815), β-actin (Cell signaling Technology, 47778) and Lamin β1 (Abcam, ab16048) were used as control for protein loading. Image J 1.4.3 was used to quantification the western results. The t-test was used for statistical analysis.
8
Temporal Expression of Circadian Genes
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The first molars were dissected from the E14.5 d, E16.5 d, E18.5 d, P.N.4 d, P.N.7 d, P.N.10 d, and P.N.15 d rats (Figures 1 , 2 ) and the E16.5d p75NTRExIII−/− and p75NTRExIII+/+ mice (Figure 4 ), fixed in 4% paraformaldehyde, decalcified with 10% EDTA, and embedded in paraffin. The 6-μm sections of tissue specimens were obtained for H.E. and immunohistochemistry staining. The primary antibodies were used in this study are as follows: rabbit anti-rat p75NTR (1:1,500; Abcam, Cambridge, MA, United States, ab245134, monoclonal), rabbit anti-rat BMAL1 (1:1,000; Abcam, Cambridge, MA, United States, ab230822, monoclonal), rabbit anti-rat CLOCK (1:1,000; Abcam, Cambridge, MA, United States, ab3517, polyclonal), rabbit anti-rat PER1 (1:500; Bioss, Beijing, China,bs-2350R, polyclonal), rabbit anti-rat CRY1 (1:500; Bioss, Beijing, China bs-11441R, polyclonal), rabbit anti-rat ALP (1:500; Bioss, Beijing, China, bs-2928R, polyclonal), rabbit anti-rat COL1 (1:500; Bioss, Beijing, China, bs-0578R, polyclonal). These specimens were treated with the DAB Detection Kit Streptavidin-Biotin (ZSGB-BIO, Beijing, China) and Hematoxylin and Eosin Staining Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocols, followed by visualization under phase-contrast microscopy.
9
Quantification of Smad3 Phosphorylation in Brown Preadipocytes
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40–50 μg of total protein from tissues or cell homogenates were used for each sample on SDS-PAGE gel. After electrophoresis, protein were transferred to PVDF membrane, blotted using specific primary and secondary antibody and detected by chemiluminescence (Supersignal; Pierce Biotechnology), as previously described49 (link). Smad3 phosphorylation in immortalized primary brown preadipocytes was assessed at 1 hour after indicated ligand treatment. The primary antibodies used were: Rev-erbα, PA5-29865 (Thermo Scientific), Bmal1, AB93806 (Abcam), UCP-1, AB3038 (Millipore); CEBPβ, sc-150, TBP, sc-204 (Santa Cruz); Smad3, 04-1035, phosphor-Smad3 (Ser 423/425), 07-1389 (Millipore).
10
Western Blot Analysis of Liver Proteins
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