To identify histone modifications, acid extraction of histone was performed as previously reported [48 (link)]. To detect other proteins, cells were extracted with lysis buffer (50 mM Tris-HCl, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1.5 mM PMSF and protease inhibitors cocktail). Equal amounts of protein were size fractionated on 6.0 to 15.0% SDS-PAGE gel. The antibodies used were anti-COX-2 (sc-19999, Santa Cruz), anti-c-Fos (ab7963, Abcam), anti-c-Jun (sc-44, Santa Cruz), anti-KMT3A (ab31358, Abcam), anti-KMT3B (17-10264, Merck Millipore), anti-KDM2A (ab31739, Abcam), anti-KDM2B (ab108276, Abcam), anti-KDM4A (ab70786, Abcam), anti-KDM4B (ab80473, Abcam), anti-NF-κB (sc-372G, Santa Cruz), anti-p300 (H-272, Santa Cruz), anti-CEBPβ (sc-150, Santa Cruz), anti-H3K4me1/2/3 (ab8895/ab7766/ab1012, Abcam), anti-H3K9me1/2/3 (ab9045/ab1220/ab8898, Abcam), anti-H3K27me1/2/3 (ab113671/ab24684/ab6002, Abcam), anti-H3K36me1/2/3 (ab9048/ab9049/ab9050, Abcam), anti-H3K79me1/2/3 (ab2886/ab3594/ab2621, Abcam), anti-H4K20me1/3 (ab9051/9053, Abcam), anti-histone H3 (ab131711, Abcam) and anti-β-actin (sc-1615, Santa Cruz).
Anti c ebpβ sc 150
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom
Anti-C/EBPβ (sc-150×) is a laboratory reagent produced by Santa Cruz Biotechnology. It is a primary antibody that specifically targets the C/EBPβ protein. C/EBPβ is a transcription factor involved in various cellular processes. The antibody can be used to detect and study the C/EBPβ protein in research applications.
Automatically generated - may contain errors
Lab products found in correlation
F7425, by Merck Group (2 mentions)
Anti pparγ sc 7273, by Santa Cruz Biotechnology (2 mentions)
Anti c ebpα, by Cell Signaling Technology (2 mentions)
Ab31739, by Abcam (1 mentions)
Ab113671, by Abcam (1 mentions)
Ab9049, by Abcam (1 mentions)
Ab7963, by Abcam (1 mentions)
Ab3594, by Abcam (1 mentions)
Ab8895, by Abcam (1 mentions)
Ab1012, by Abcam (1 mentions)
Ab7766, by Abcam (1 mentions)
Ab9045, by Abcam (1 mentions)
Ab2886, by Abcam (1 mentions)
Ab2621, by Abcam (1 mentions)
Ab9048, by Abcam (1 mentions)
Ab8898, by Abcam (1 mentions)
Ab1220, by Abcam (1 mentions)
Ab9050, by Abcam (1 mentions)
Ab6002, by Abcam (1 mentions)
Ab24684, by Abcam (1 mentions)
9 protocols using anti c ebpβ sc 150
1
Comprehensive Protein Detection and Histone Modification Analysis
2
Protein Immunoprecipitation and Western Blot
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The cells were lysed in 1 ml lysis-buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100, 2 mM EDTA, 25 mM glycerophosphate, 100 mM NaF, 200 mM Na3VO4, 1 mg/ml leupeptin, 1 mg/ml aprotinin, and 1 mM PMSF). According to the manufacturer’s protocol, the insoluble material in cell lysates was removed via centrifugation, and the recovered supernatant was immunoprecipitated with Protein G-coated magnetic Dynabeads (Invitrogen) and 10 µg of each of the following antibodies separately: anti-JMJD3 (ab85392, Abcam); anti-C/EBPβ (sc-150×, Santa Cruz); anti-FLAG (F7425, Sigma), and control IgG (ab172730, Abcam). To be prepared for western blotting analysis, firstly the immunoprecipitates were washed four times with 1 ml lysis-buffer, then resuspended in 50 µl 2× Laemmli sample buffer (125 mM Tris pH 6.8, 4% SDS, 25% glycerol, 0.1% bromophenol blue, 5% β-mercaptoethanol), and finally boiled at 95 °C for 10 min. Control (INPUT) was made by 50 µl cell lysates processed for all steps except for the incubation with Protein G-coated magnetic Dynabeads and antibodies. The uncropped scans of blots are shown in the Supplementary Fig. 7 .
3
Western Blot Analysis of Protein Expression
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Protein samples were separated by SDS-PAGE and transferred to an Immobilon-P polyvinylidene fluoride transfer membrane (Millipore). After blocking, the membrane was incubated with a primary antibody overnight at 4°C, followed by incubation with the horseradish peroxidase-conjugated secondary antibody (HRP-conjugated goat anti-mouse IgG, Santa Cruz Biotechnology; HRP-conjugated goat anti-rabbit IgG, Novus Biologicals). Primary antibodies used in this study were anti-UCP1 (an antiserum from a rabbit immunized with the rat UCP1 purified from BAT in cold-exposed rats, as described previously81 (link)), anti-β-Actin (4967; Cell Signaling), anti-HMGCR (ab174830; Abcam), anti-RB (ab181616; Abcam), anti-C/EBPβ (sc-150; Santa Cruz Biotechnology), anti-C/EBPδ (sc-636; Santa Cruz Biotechnology), anti-α-Tubulin (2144; Cell Signaling), anti-Histone H3 (3638; Cell Signaling), anti-Caspase 3 (GTX110543; GeneTex), and anti-Cleaved Caspase 3 (Asp175) (9661; Cell Signaling). Proteins were visualized by chemiluminescence using an Immobilon Western Chemiluminescent HRP Substrate (Millipore). The chemiluminescent signal was detected using an ImageQuant LAS4000 (GE Healthcare) apparatus. The intensity of Western blot bands was quantified with ImageJ software (National Institutes of Health).
4
Western Blot Analysis of Protein Expression
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Total cell lysates or protein extracts were equally loaded on 8–12% SDS-polyacrylamide gel for running and then transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare-Amersham Biosciences, UK). After blocking with 5% non-fat milk in Tris buffered saline with Tween 20 (TBST), the membranes were incubated for 2 h or overnight with the primary antibodies. After staining with horseradish peroxidase (HRP)-linked secondary antibodies, signal detection was performed using a chemiluminescence phototope-HRP Kit (Millipore). The band intensity was estimated using the Photoshop Software. Densitometric analysis was performed using ImageJ software (NIH, Bethesda, MD). The following antibodies were used: anti-JMJD3 (ab154126, Abcam, 1:250), anti-C/EBPβ (sc-150×, Santa Cruz, 1:1000), anti-C/EBPα (2843, Cell Signaling, 1:1000), anti-RIPK3 (ab56164, Abcam, 1:1000), anti-JUNB (3753S, Cell Signaling, 1:1000), anti-Flag (F7425, Sigma, 1:5000) and anti-Myc (9B11, Cell Signaling, 1:1000), and anti-β-actin (A5316, Sigma, 1:5000).
5
Immunodetection of Adipogenic Markers
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Anti‐laminin (L9393; 1:400) antibody was purchased from Sigma‐Aldrich and secondary antibody Alexa Fluor 488 (A11029; 1:800) was purchased from Invitrogen. Anti‐PDGFRα (#3174), anti‐perilipin (#9349), anti‐FABP4 (#3544) and anti‐C/EBPα (#8178) primary antibodies were purchased from Cell Signalling and used at 1:500 dilution. Anti‐PPARγ (sc‐7273) and anti‐C/EBPβ (sc‐150) primary antibodies were purchased from Santa Cruz and used at 1:200 dilution. Anti‐mouse (sc‐2005) and anti‐rabbit (sc‐2004) HRP‐conjugated secondary antibodies were purchased from Santa Cruz and used at 1:4000 dilution.
6
Transcription Factor Expression Analysis
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RNA extraction was processed according to the RNeasy (QIAGEN) protocol. After genomic DNA degradation with the RNase-Free DNase kit (QIAGEN), reverse transcription was performed with the Superscript III (Invitrogen). For Srebf1, Klf4, Klf5, Krox20, C/EBPδ, C/EBPβ, Prep1, Glut4 expression analysis, cDNAs were subjected to qRT-PCR on a Roche LightCycler480 (Roche) using predesigned primers (RealTime ready assays, Roche). Sequences are available upon request. Results were normalized to Gapdh gene expression.
Antibodies used were anti-Prep1 (Santa Cruz sc-25282), anti-C/EBPα (D56F10 Cell signaling Technology #8178), anti-C/EBPβ (sc-150, Santa Cruz Biotechnology, Santa Cruz, USA), anti-phospho-C/EBPβ (Thr235) (Cell Signaling Technology #3084), anti-C/EBPδ (sc-151, Santa Cruz Biotechnology, Santa Cruz, USA), anti-Pparγ (81B8, Cell signaling Technology #2443), anti-pIrs1 (Tyr941) (07-848-I Millipore), anti-pAkt (Ser473) (D9E Cell signaling Technology #4060), anti-pAkt (Thr308) (D25E6 Cell signaling Technology #13038), anti-Klf5 (Abcam ab24331), anti-Vinculin (Sigma V9131).
Antibodies used were anti-Prep1 (Santa Cruz sc-25282), anti-C/EBPα (D56F10 Cell signaling Technology #8178), anti-C/EBPβ (sc-150, Santa Cruz Biotechnology, Santa Cruz, USA), anti-phospho-C/EBPβ (Thr235) (Cell Signaling Technology #3084), anti-C/EBPδ (sc-151, Santa Cruz Biotechnology, Santa Cruz, USA), anti-Pparγ (81B8, Cell signaling Technology #2443), anti-pIrs1 (Tyr941) (07-848-I Millipore), anti-pAkt (Ser473) (D9E Cell signaling Technology #4060), anti-pAkt (Thr308) (D25E6 Cell signaling Technology #13038), anti-Klf5 (Abcam ab24331), anti-Vinculin (Sigma V9131).
7
ChIP-qPCR Analysis of Neutrophils
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ChIP experiments were performed exactly as previously described8 (link). Briefly, nuclear extracts from 5 × 106 or 107 neutrophils (for ChIP targeting, respectively, H3K27Ac or STAT1, IRF1, C/EBPβ and PolII) were immunoprecipitated with 1 μl anti-H3K27Ac (ab4729) (Abcam, Cambridge, United Kingdom), 25 μl anti-STAT1 (sc-346), 25 μl anti-IRF1 (sc-497), 20 μl anti- C/EBPβ (sc-150), 20 μl anti-PolII (sc-899) (Santa Cruz Biotechnology). To establish the background levels of ChIP experiments, the precipitation signal was quantified also at the promoter of prolactin (PRL) since it is completely silent in myeloid cells. The coimmunoprecipitated material was subjected to qPCR analysis using the following promoter specific primers (purchased from Life Technologies): IL-6 forward: 5′-TAGCCTCAATGACGACCTAAG-3′; IL-6 reverse: 5′-GTGGGGCTGATTGGAAACCT-3′; IFIT1 forward: 5′-GGCAGCAATGGACTGATGTTC-3′; IFIT1 reverse: 5′-GGAAACCGAAAGGGGAAAGTG-3′; and PRL forward: 5′-AGGGAAACGAATGCCTGATT-3′; PRL reverse: 5′-GCAGGAAACACACTTCACCA-3′. Data from qPCR were expressed as percentage over input DNA and are displayed as means ± SEM.
8
Antibody panel for adipogenesis
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Anti-PDGFRα (#3174), anti-perilipin (#9349), anti-FABP4 (#3544) and anti-C/EBPα (#8178) primary antibodies were purchased from Cell Signaling and used at 1:500. Anti-PPARγ (sc-7273; 1:200), anti-C/EBPβ (sc-150; 1:200) anti-TNFα (sc-52746; 1:200), and anti-β-actin (sc-81178; 1:4000) primary antibodies were purchased from Santa Cruz. Anti-mouse (sc-2005) and anti-rabbit (sc-2004) HRP-conjugated secondary antibodies were purchased from Santa Cruz and used at 1:4000.
9
Antibody Sources and Oligonucleotide Providers
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Reagents NHERF-1 antibody was a gift from Dr. E. J. Weinman (University of Maryland) [1] . Anti-Sp3 (sc-644x), anti-Sp1 (sc-14027x), and anti-C/EBPβ (sc-150) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). DNA oligonucleotides were ordered from Integrated DNA Technologies, Inc. (Redwood City, CA).
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