Western blot analysis was performed using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis system. Protein samples (20 µg) were resuspended in reduced sample buffer, electrophoresed on a 7.5-10% Tris gel with Tris running buffer, blotted onto PVDF membranes, and sequentially probed with primary antibodies against RUNX2 (Cat. no. 12556; 1:1,000; Cell Signaling Technology), OCN (Cat. no. sc-365797; 1:1,000; Santa Cruz Biotechnology, Inc.), eukaryotic translation initiation factor 4E-binding protein 1 (4E/BP1; Thr37/46; Cat. no. 9644; 1:1,000; Cell Signaling Technology), phospho-S6 ribosomal protein (P-S6; S235/S236; Cat. no. sc-293143; 1:1,000), PPARγ (Cat. no. sc-7273; 1:1,000) (both from Santa Cruz Biotechnology, Inc.), C/EBPβ (Cat. no. 3802; 1:2,000), Sca-1 (Cat. no. 9664; 1:4,000), CD29 (Cat. no. 4706; 1:2,000), CD45 (Cat. no. 13917; 1:6,000), CD11b (Cat. no. 14271; 1:3,000) and β-actin (Cat. no. 3700; 1:2,500) (all from Cell Signaling Technology). Horseradish peroxidase-conjugated goat anti-rabbit (Cat. no. SAB4600223; 1:1,000) or anti-mouse (Cat. no. SAB4600004; 1:1,000) antibodies (both from Sigma) were added, and secondary antibodies were detected using enhanced chemiluminescence (ECL Plus; General Electric Healthcare, Milwaukee, WI, USA).
C ebpβ
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom
C/EBPβ is a transcription factor that plays a role in regulating gene expression. It is involved in various cellular processes, including cell differentiation, proliferation, and immune response.
Automatically generated - may contain errors
Lab products found in correlation
C ebpα, by Santa Cruz Biotechnology (12 mentions)
Pparγ, by Santa Cruz Biotechnology (9 mentions)
β actin, by Santa Cruz Biotechnology (7 mentions)
β actin, by Merck Group (5 mentions)
Dexamethasone (dex), by Merck Group (5 mentions)
α tubulin, by Santa Cruz Biotechnology (3 mentions)
P erk, by Cell Signaling Technology (3 mentions)
C ebpδ, by Santa Cruz Biotechnology (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Gapdh, by Proteintech (2 mentions)
Loading buffer reagents, by Santa Cruz Biotechnology (2 mentions)
Ecl western blotting detection system, by Cytiva (2 mentions)
Hyperfilm ecl, by Cytiva (2 mentions)
β tubulin, by Merck Group (2 mentions)
Prdm16, by Santa Cruz Biotechnology (2 mentions)
Hsp90, by Cell Signaling Technology (2 mentions)
Protease inhibitor and phosphatase inhibitor, by Roche (2 mentions)
C ebp γ, by Santa Cruz Biotechnology (2 mentions)
65 protocols using c ebpβ
1
Comprehensive Western Blot Analysis
2
ChIP-seq analysis of CREB and C/EBPβ
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PBMC were plated at 2 million cells per ml in RPMI containing 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. These cells were then treated with 10 nM ET (10 nM EF and 10 nM PA) or 500 μM 6MB-cAMP for 4.5 h and then isolated by centrifugation followed by snap freezing in liquid N2 as recommended by Active Motif. For each condition, 60 million PBMC were prepared from 3 donors (20 million PBMC per donor). ChIP was performed by Active Motif with antibodies validated for ChIP against CREB (Cell Signaling Tech; catalog number 9197) or against C/EBP β (Santa Cruz Biotechnology, catalog number sc-150). After ChIP DNA libraries were constructed, 75-nt sequence reads were generated by Illumina sequencing.
3
Western Blotting Procedure for Protein Analysis
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The cells were lysed in ice-cold radioimmunoprecipitation assay lysis buffer (Cell signalling Technology, MA, USA) containing a protease inhibitor cocktail (Roche, Basel, Switzerland) and phosphatase inhibitor (Roche). Protein (40 μg) from each sample was electrophoresed in a 10% sodium dodecyl sulphate (SDS)–polyacrylamide gel and transferred to a nitrocellulose blot. After blocking, the blot was incubated with antibodies against FHL2 (1:1000; Proteintech), FHL2 (1:1000; Abcam, Cambridge, UK), AR (1:400; Santa Cruz Biotechnology, CA, USA), AR (1:1000; Cell signalling Technology), C/EBPβ (1:400; Santa Cruz Biotechnology), COX2 (1:1000; Proteintech), HAS2 (1:1000; Abcam), total ERK1/2, and ERK1/2 phosphorylated at Thr202/Tyr204 (1:2000; Cell signalling Technology) overnight at 4 °C, followed by incubation with secondary antibodies (1:5000; SAB, Maryland, USA). An enhanced chemiluminescence detection system (Millipore) was used to detect protein bands. The same blot was probed for GAPDH (1:2000; Proteintech) as an internal loading control. The bands were visualised using the G-Box iChemi Chemiluminescence Image Capture System (Syngene, MD, USA).
4
Quantitative Western Blotting Protocol
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Western blotting was performed as reported previously (80 (link), 81 (link)). In brief, whole-cell lysates were prepared using loading buffer reagents (Santa Cruz Biotechnology, Inc) without trypsin treatment. Equal amounts of total protein were electrophoresed on a 10% SDS–polyacrylamide gel. The proteins were transferred to polyvinylidene difluoride membranes (ATTO). The membranes were blocked with blocking solution (5% skimmed milk with 0.1% Tween-20 dissolved in Tris-buffered saline [pH 7.5]), incubated with the first antibody for PGC-1α (Calbiochem), C/EBPβ (Santa Cruz Biotechnology), and β-tubulin (Sigma), which were diluted in blocking solution, incubated with the peroxidase-conjugated second antibody diluted in blocking solution, visualized with the ECL-Western blotting detection system (Amersham) according to the manufacturer's protocol, and used to expose hyperfilm-ECL (Amersham). To reuse the blot, the membranes were stripped in Restore Western stripping buffer (Pierce). Western blot bands were quantified by ImageJ (U. S. National Institutes of Health), and the quantification values of three independent experiments are provided in Table S2 of the revised manuscript. The uncropped images of immunoblots are shown in Fig. S1 .
5
Western Blotting of Key Protein Markers
6
Protein Expression Analysis in Cell Lysates
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Cells were lysed by using a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) sample loading buffer. The lysates were subjected to PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked with 5% nonfat dried milk and probed with the following specific primary antibodies: C/EBP‐β, sterol regulatory element binding protein (SREBP)‐1 (2A4), and fatty acid synthase (FASN) (A‐5; Santa Cruz); ATG5 (Proteintech, IL); and mammalian target of rapamycin (mTOR; 7C10) and phosphorylated‐mTOR (p‐mTOR; D9C2) (Cell Signaling Technology, Danvers, MA). After washing, the blots were incubated with secondary antibody for 1 hour. Proteins were detected by using an enhanced chemiluminescence western blot substrate (Pierce; ThermoFisher Scientific). Membranes were reprobed with antibody to actin (Santa Cruz) as an internal control for normalization of the protein load. ImageJ software (National Institutes of Health [NIH]) was used for densitometric scanning of western blot images.
7
Protein Expression Analysis in Heart Tissues
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Total proteins isolated from the heart tissues were size fractionated using SDS-PAGE and transferred onto Immobilon-P membranes (Millipore, Billerica, MA). The blotted membranes were incubated with antibodies against p-ERK, t-ERK, p-ErbB2, p-ErbB4, LC3b (Cell Signaling Technology, Beverly, MA), β1-AR (beta-1 adrenergic receptor; Abcam, Cambridge, MA), C/EBPβ, and AT1-R, (Santa Cruz Biotechnology Inc, Santa Cruz, CA) and with an HRP-conjugated secondary antibody (1:5000, KangChen Biotechnology, Shanghai, China). Either GAPDH or t-ERK was used as an internal control. The proteins were visualized using an ECL Western blotting detection system (GE Healthcare, catalog number RPN2106). The relative intensities of the protein bands were analyzed through densitometry with a gel documentation system using LAS-300 image analysis software. All experiments were repeated at least 3 times.
8
ChIP-seq and ChIP-qPCR Protocols for RNAPII, CEBPβ, H3K27Ac, CTCF
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ChIP was performed by standard protocols for ChIP-qPCR and ChIP (12 (link),26 (link)). Antibodies were specific for RNAPII (Abcam ab76123 for ChIP-qPCR or Cell Signaling Technology 14958S for ChIP-seq), CEBPβ (Santa Cruz sc-7962), H3K27Ac (Millipore 07-360), CTCF (Millipore 07-729) or rabbit IgG (Millipore 12-370). All ChIP-seq experiments were performed twice and irreproducible discovery rate (IDR) data are shown. Raw reads were processed using the AQUAS Transcription Factor and Histone ChIP-Seq processing pipeline (https://github.com/ENCODE-DCC/chip-seq-pipeline2 ) using the ENCODE (phase-3) guidelines on the hg19 reference genome. This includes mapping using BWA and peak calling with MACS2. Primer sequences used for qPCR are shown in Supplementary Table S2 .
9
Western Blot Analysis of Adipogenesis Regulators
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Homogenized cells were ice-cold lysed by ice-cold RIPA buffer [50 mM Tris-Cl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 0.5% Sodium deoxycholate, 1% Triton X-100, 50 mM β-glycerophosphate, 50 mM NaF, and 1 mM Na3VO4] containing protease inhibitor (Roche, Switzerland). After centrifugation at 13,000 rpm for 15 min, supernatants were transferred to a new tube. Protein concentrations were measured using the Bradford assay (Bio-Rad, USA). Western blot analysis was performed using standard protocols (41 (link)). The following primary antibodies were used: PPARγ (2435s, CST), C/EBPα (2295, CST), C/EBPβ (sc7962, Santa Cruz), FABP4 (2120s, CST), Akt (9272s, CST), p-Akt (9271s, CST), AMPKα (2532s, CST), p-AMPKα (2535s, CST), β-Catenin (06-734, Sigma), p38 (9212s, CST), p-p38 (9215s, CST), ERK (9102s, CST), p-ERK (9106s, CST), JNK (9252s, CST), p-JNK (9251s, CST), CHOP (2895s, CST), and HSP90 (sc-13119, Santa Cruz). Specific antibody signals were detected by horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit IgG antibody (Santa Cruz) and detected with an enhanced chemiluminescence system (Fusion Solo S, Vilber Lourmat, France). The relative amounts of each protein band were quantified with the ImageJ software.
10
Protein Expression Analysis in Cells
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Cells were harvested by scrapping with iced cold PBS and lysed directly in RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 5 mM EDTA (pH 8.0), and 1 mM EGTA and supplemented with protease and phosphatase inhibitors at 4°C for 20 min and then centrifuged with 12000 rpm at 4°C for 30 min and total protein content was determined by Bradford assay. Equal amounts of total proteins were separated by SDS-polyacrylamide gel electrophoresis. Immunoblotting was performed using primary antibodies against Src, phosphorylated-Src (p-Src), paxillin, and phosphorylated-paxillin (p-PXN) (Cell Signaling Technology, Beverly, MA) and ERK1/2, phosphorylated-ERK1/2 (p-ERK1/2), c-Fos, C/EBP-β, phosphorylated-C/EBP-β (p-C/EBP-β), FAK, and phosphorylated-FAK (p-FAK) (Santa Cruz, CA, USA). The image was investigated using an ECL-Plus detection kit (PerkinElmer Life Sciences, Inc., Boston, MA, USA).
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