DNA and associated proteins on chromatin in cultured cells were crosslinked by 1% formaldehyde for 15 min at 37 °C. Cells were then scraped and collected in cellular lysis buffer (5 mM Pipes, 85 mM KCl, 0.5% NP-40, and protease inhibitors). Cytoplasmic lysates were discarded and nuclear components were resuspended in nuclear lysis buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.2% SDS, and protease inhibitors), and sonicated for 10 min (Covaris). Approximately 4 mg of CEBPA (Cat# sc-365318, Santa cruz), CEBPD (Cat# sc-365546, Santa Cruz Biotechnology) or control IgG (Cat# ab6715, Abcam) were incubated with 25 mL of protein G magnetic beads for 6 h at 4 °C, and then incubated with 100 mg of cleared chromatin overnight at 4 °C. After three washes, immunoprecipitated material was eluted at 55 °C for 1 h with 10 µg/mL proteinase K, and then decrosslinked at 65 °C for 4 h. The primer sequences used for ChIP-qPCR were listed in Supplementary Table S4 .
Cebpa
Manufactured by Santa Cruz Biotechnology
Sourced in United Kingdom, United States
CEBPA is a protein that plays a role in the regulation of genes involved in cell growth and differentiation. It functions as a transcriptional activator and is expressed in several tissues, including the liver, lung, and adipose tissue. The CEBPA lab equipment provides researchers with a tool to study the expression and function of this important protein.
Automatically generated - may contain errors
Lab products found in correlation
Cebpd, by Santa Cruz Biotechnology (2 mentions)
Control igg, by Abcam (1 mentions)
Truseq dna sample kit, by Illumina (1 mentions)
Rabbit igg, by Merck Group (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
C fos, by Santa Cruz Biotechnology (1 mentions)
Mmp 9, by Santa Cruz Biotechnology (1 mentions)
Cathepsin k, by Santa Cruz Biotechnology (1 mentions)
Total nf kb p65, by Cell Signaling Technology (1 mentions)
H3k4me3, by Merck Group (1 mentions)
Nfatc1, by Santa Cruz Biotechnology (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Phosphorylated akt, by Cell Signaling Technology (1 mentions)
H3k27me3, by Merck Group (1 mentions)
H3k4me1, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Spectra x light engine light source, by Lumencor (1 mentions)
Cfi plan apo vc 20, by Nikon (1 mentions)
Alexa fluor 647, by Abcam (1 mentions)
Donkey antimouse alexafluor 488, by Abcam (1 mentions)
7 protocols using cebpa
1
Chromatin Immunoprecipitation (ChIP) Protocol
2
ChIP-Seq Analysis of Transcription Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP was performed as previously described29 (link) using the following antibodies: PU.1 (Santa Cruz Biotechnologies, sc352x), CEBPA (Santa Cruz Biotechnologies, sc61x), H3AcK27 (Abcam, Cambridge, UK; ab4729) and rabbit IgG (Sigma, I5006). Library construction was performed using the Illumina TruSeq DNA Sample Kit (Illumina, Cambridge, UK) according to the manufacturer's instructions. Sequencing was performed on the Illumina HiSeq 2000 platform. Reads were mapped to the mm10 mouse reference genome using Bowtie2.30 (link) Mapped reads were converted to density plots and displayed as UCSC genome browser custom tracks, and peaks called using MACS2.31 (link)Using BEDTools,32 (link) ChIP-Seq peak coordinates were combined between PU.1− and PU.1+ conditions for each TF ChIP, and peaks overlapping by at least 1 bp were merged. Coverage scores were counted using the intersectBed function for each merged peak region, and then normalised for peak length and total read counts (per 1 million reads). For H3K27Ac, read coverage regions were extended to 800 bp, and then normalised as above.
3
Comprehensive Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed as previously described 33 (link). Antibodies used are cathepsin k (Santa Cruz, cat# sc-48353), Mmp9 (Santa Cruz, cat# sc-6840), Gapdh (Santa Cruz, cat# sc-25778), phosphorylated Nfkb p65 (Cell Signaling, cat# 3033S), total Nfkb p65 (Cell Signaling, cat# 8242S), phosphorylated Akt (Cell Signaling, cat# 2965S), total Akt (Cell Signaling, cat# 2938S), H3 (Millipore/Sigma, cat#06-755), H3K36me3 (Millipore/Sigma, cat#05-801), H3K18ac(Millipore/Sigma, cat#07-354), H3K27me3 (Millipore/Sigma, cat#07-449), H3k4me1 (Millipore/Sigma, cat#07-436), H3k4me3 (Millipore/Sigma, cat#07-473), NFATC1 (Santa Cruz sc-7294), CEBPa (Santa Cruz sc-61), PU.1 (Santa Cruz sc-352),ATP6i (Santa Cruz sc-162300), c-Fos (Santa Cruz sc-52), Beta-Tubulin (E7; DSHB).
4
Multiparameter Immunofluorescence Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell samples were washed with PBS, fixed in 4% paraformaldehyde, blocked in 2% bovine serum albumin (BSA), permeabilized with 0.5% (vol/vol) Triton X-100 in PBS for 20 min, immersed in 2% BSA for 1 h, and rinsed in PBS. Staining using the primary antibodies targeting ATF4 and RUNX2 (Abcam), CEBPA (Santa Cruz), TPM1/2 (TM311, Sigma-Aldrich), and YAP1 (Proteintech), and the secondary antibodies donkey anti-mouse (Alexa Fluor 488; 1:100; Abcam), donkey anti-rabbit (Alexa Fluor 594), donkey anti-mouse (Alexa Fluor 647; 1:100; Abcam), and donkey anti-rabbit (Alexa Fluor 647; 1:100; Abcam) was performed according to manufacturer’s protocols. Samples were stained with 1 µg/mL−1 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), dissolved in PBS, and immersed for 20 min and in 165 nM Phalloidin-iFluor 555 dissolved in PBS and immersed for 30 min. Lipid droplet staining was performed by immersing cells in 0.1 μg/mL−1 Nile-red (Sigma) dissolved in distilled water (DW) and immersed for 5 min. Immunofluorescence imaging was performed using a NIKON Ti-E inverted microscope equipped with an sCMOS iXon3 camera (Anodr) and a Spectra X light engine light source (Lumencor). A CFI Apo TIRF 60× Oil (Nikon) and a CFI Plan Apo VC 20× (Nikon) objective were used. Cell and nucleus projected areas were segmented and quantified using a custom-designed MATLAB code.
5
Immunoblotting Analysis of Cellular Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates and supernatants were resolved by electrophoresis, transferred to a polyvinylidene fluoride membrane, and probed with antibodies against β-tubulin (Cat# 2128, Cell Signaling Technology), iNOS (Cat# ab178945, Abcam), CD36 (Cat# ab252922, Abcam), CEBPA (Cat# sc-365318, Santa cruz), CEBPD (Cat# sc-365546, Santa Cruz Biotechnology), MIF (Cat# ab187064, Abcam), p-p38 (Cat# ab195049, Abcam), p65 (Cat# ab32536, Abcam), SOX2 (Cat# ab92494, Abcam), OCT4 (Cat# ab181557, Abcam), STAT3 (Cat# ab68153, Abcam), or HLA-DRA (Cat# ab92511, Abcam). The antibodies were listed in the Supplementary Table S5 .
6
High-fat Diet Effects on Metabolic Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
High-fat diet (60% kcal from fat) was purchased from Research Diets Inc (New Brunswick, NJ, USA). Detection kits of serum triglyceride (TG), total cholesterol (TC), and LDL-cholesterol (LDL-C) were purchased from Beckman Coulter Inc (Brea, Cal, USA). Kits of fasting blood glucose and insulin were supplied by Allmedicus (Gyeonggi-do, Republic of Korea) and Shibayagi Co. Ltd (Gumma Pref, Japan), respectively. Dulbecco's modified eagle's medium, newborn calf serum, and fetal bovine serum were purchased from Gibco (BRL, NY, USA). Oil Red O (ORO), compound C {6-[4-(2-piperidin-1-yl-etoxy)-phenyl]-3-pyridin-4-yl-pyrrazolo [1, 5-α] pyrimidine}, isobutylmethylxanthine, insulin, and dexamethasone were obtained from Sigma (St. Louis, MO, USA). Primary antibodies against AMPK, p-AMPK Thr172 , PPAR-γ, FAS, ACC, and p-ACC Ser79 were obtained from Cell Signaling Technology (Beverly, MA, USA). C/EBPa, SREBP-1c, and ß-actin were also purchased from Santa Cruz Biotechnology Inc (Dallas, Tex, USA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Jackson ImmunoResearch Inc (West Grove, PA, USA). A Western blot enhanced chemiluminescence kit was purchased from IMGENEX (San Diego, CA, USA).
7
Western Blot Analysis of Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells collected in lysis buffer were lyzed by three freezeethaw cycles, and proteins were denatured by incubation with 4x Laemmli buffer at 95 C for 10 min. After separation by PAGE, proteins were blotted onto a PVDF membrane using a semi-dry blotting apparatus of Bio-Rad. The proteins of interest were detected using primary antibodies against b-Actin, Pparg, C/ebpb and C/ebpa from Santa Cruz and appropriate secondary HRP-conjugated antibodies (Dianova and SigmaeAldrich). After ECL detection (Pierce reagent), the images were captured using a Fusion FX7 apparatus (Vilber Lourmat).
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!