For protein extraction, the cells were washed twice with ice-cold 1 × PBS and lysed in 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, supplemented with protease and phosphatase inhibitors followed by sonication. Equal amounts of total protein were separated by SDS–PAGE (#456-1094, BIO-RAD), transferred to a PVDF membrane using Trans-Blot Turbo System (#170-4273, BIO-RAD) following the manufacturer’s protocol. The following antibodies were used: C/EBPβ (C19) and SBDS (S-15) from Santa Cruz Biotechnology; phospho-p70S6K (Thr389) (108D2), p70S6K (#9202), Hamartin/TSC1 (D43E2) (#6935), phospho-4E-BP1 (Thr37/46) (#9459), 4E-BP1 (#9452), 4E-BP2 (#2845), eIF4E (#9742), Phospho-eIF2α (Ser51) (#9721), eIF2α (#9722) and PKR (#3072) from Cell Signaling; DENR (#10656-1-AP) from ProteintechTM and β-actin (clone C4) (#691001) from MP Biomedicals. HRP-conjugated secondary antibodies were purchased from Amersham Life Technologies. The bands were visualized by chemiluminescence (ECL, Amersham Life Technologies) using ImageQuant LAS 4000 mini imaging machine (GE Healthcare Bioscience AB) and the supplied software was used for the quantification of the bands.
C ebpβ c 19
Manufactured by Santa Cruz Biotechnology
Sourced in United States
C/EBPβ (C-19) is a recombinant protein that corresponds to the C-terminal amino acids 220-296 of the human C/EBPβ transcription factor. C/EBPβ is a member of the CCAAT/enhancer binding protein family and functions as a basic leucine zipper (bZIP) domain transcription factor involved in the regulation of gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot turbo system, by Bio-Rad (3 mentions)
Las 4000, by Cytiva (2 mentions)
C ebpδ c 22, by Santa Cruz Biotechnology (2 mentions)
Phospho eif2α ser51, by Cell Signaling Technology (1 mentions)
Phospho 4e bp1 thr37 46, by Cell Signaling Technology (1 mentions)
β actin clone c4, by MP Biomedicals (1 mentions)
4e bp2, by Cell Signaling Technology (1 mentions)
Eif4e, by Cell Signaling Technology (1 mentions)
Eif2α, by Cell Signaling Technology (1 mentions)
4e bp1, by Cell Signaling Technology (1 mentions)
Las 4000 luminescent image analyzer, by Fujifilm (1 mentions)
Hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Protease and phosphatase inhibitors, by Santa Cruz Biotechnology (1 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
Phospho smad2 3, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Normal rabbit igg sc 2027, by Santa Cruz Biotechnology (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
5 protocols using c ebpβ c 19
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Skeletal Muscle Proteins
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Protein extracts were harvested with protease and phosphatase inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and were analyzed using the following antibodies: C/EBPβ (C-19, Santa Cruz Biotechnology), Smad2/3 (Cell Signaling Technology, Danvers, MA, USA), myogenin, Pax7, and myosin heavy chain (MF-20) primary antibodies from DSHB (Iowa City, IA, USA); and phospho Smad2/3 (Abcam, Cambridge, U.K.). β-Actin (Sigma-Aldrich, St-Louis, MO, USA) was used as a loading control. HRP-conjugated secondary antibodies were from GE Healthcare (Buckinghamshire, U.K.). Chemiluminescence images were captured using the Luminescent Image Analyzer LAS-4000 (Fujifilm Life Science, Tokyo, Japan), and quantifications were done using ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij/ , 1997–2014).
3
Western Blot Analysis of C/EBP Proteins
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Total protein lysates were prepared by lysing fresh cell pellets with 0.5 M NaOH with subsequent neutralization with 0.5 M HCl. The samples were sonicated, mixed with SDS sample buffer and glycerol, and heated (3 min at 95°C). After centrifugation, the protein lysates were separated by electrophoresis and transferred to a nitrocellulose membrane using a Trans-Blot Turbo System (Bio-Rad). Protein signals were detected after incubation with antibodies against C/EBPα (14AA; Santa Cruz Biotechnology), C/EBPβ (C-19; Santa Cruz Biotechnology), C/EBPδ (C-22; Santa Cruz Biotechnology), C/EBPε (NBP1-85446; Novus Biologicals), Flag-M2 (F-1804; Sigma-Aldrich), and GAPDH (ab9484; Abcam).
4
Immunohistochemical Analysis of Growth Plate
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Tissue samples of growth plate were obtained from mouse embryos (E16.5). For immunoperoxidase method, Vectastain Elite ABC kit (Vector Laboratories; Burlingame, CA) was used. Deparaffinized sections (3 µm thickness) were subjected to antigen retrieval by microwaving in 10 mM citrate buffer (sodium citrate, pH 6.0) for 20 minutes. Endogenous peroxidase activity was blocked by incubation in 3% H2O2 in methanol for 30 minutes. The specimens were placed in blocking reagent for 30 minutes and incubated overnight at 4°C with the following primary antibodies: C/EBPβ (C-19; Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1∶500, RUNX2 (AP7735a; Abgent, San Diego, CA) diluted 1∶200, Ihh (C-15; Santa Cruz Biotechnology), or normal rabbit IgG (sc-2027; Santa Cruz Biotechnology) diluted 1∶1000. The samples were further incubated with secondary antibodies for 30 minutes and then a colorimetric reaction was carried out with 3,3′-diaminobenzidine and 0.02% H2O2, followed by counterstaining with hematoxylin. For immunofluorescent staining, Alexa Fluor 568 (Invitrogen, Carlsbad, CA) were used as a secondary antibody and mounted with VECTASHIELD Mounting Medium with DAPI (Vector Laboratories).
5
Protein Extraction and Western Blotting
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Total protein lysates were prepared by incubation of snap-frozen cell pellets with lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 10% glycerol, and proteinase inhibitor cocktail [Roche] in H2O) for 30 min on ice. After centrifugation protein concentration was measured in supernatants using Pierce Reagent (Thermo Scientific). Protein lysates were subjected to electrophoresis and transferred to a nitrocellulose membrane on the Trans-Blot Turbo System (Bio-Rad). Specific protein signals were detected by incubation with antibodies against C/EBPα (14AA, Santa Cruz Biotechnology), C/EBPβ (C-19, Santa Cruz), C/EBPδ (C-22, Santa Cruz), C/EBPε (C-22, Santa Cruz), GFP (7.1/13.1, Roche), and ACTB (AC-15, Sigma).
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