Primary antibodies against β-actin, FAK (Focal adhesion kinase), p-397Tyr-FAK, IKKα (I-kappaB kinase α), IKKβ (I-kappaB kinase β), β-catenin, total-p53, p21, and MnSOD were purchased from Santa Cruz Biotechnology (Santa Cruz, Dallas, TX, USA). Primary antibodies against UCP2, NF-κB p65, p-536Ser-NF-κB p65, S6 ribosomal protein and cleaved caspase 3, were purchased from Cell Signaling Technology (Boston, MA, USA). Primary antibodies against wild type p53 were purchased from MilliporeSigma (Burlington, MA, USA). On a 10% SDS–PAGE gel, 20 μg total protein was electrophoresed, transferred onto a polyvinylidene fluoride membranes, blocked, incubated with a primary antibody and then with a horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA). Immunoreactive bands were visualized using a chemiluminescence solution (Genesee Scientific, El Cajon, CA, USA). Experiments were repeated three times. β-actin was employed as an endogenous control. The pixel densities of the protein bands were quantified using ImageJ.
Total p53
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Total p53 is a lab equipment product that measures the total amount of the p53 protein in a sample. p53 is a critical regulator of cellular processes and is commonly studied in various research applications. This product provides a quantitative assessment of p53 levels without interpretation of its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescence solution, by Genesee Scientific (1 mentions)
Mnsod, by Santa Cruz Biotechnology (1 mentions)
S6 ribosomal protein, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
β catenin, by Santa Cruz Biotechnology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Iblot transfer stack, by Thermo Fisher Scientific (1 mentions)
Nupage precast gel, by Thermo Fisher Scientific (1 mentions)
Anti human gapdh, by Merck Group (1 mentions)
Anti mouse and anti rabbit secondary antibodies, by GE Healthcare (1 mentions)
Ampkα, by Cell Signaling Technology (1 mentions)
Tween 20, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Hsp70, by Santa Cruz Biotechnology (1 mentions)
Ab1790, by Abcam (1 mentions)
Ab2175, by Abcam (1 mentions)
Ab9050, by Abcam (1 mentions)
Ab38497, by Abcam (1 mentions)
Ab1791, by Abcam (1 mentions)
5 protocols using total p53
1
Western Blot Analysis of Cellular Proteins
2
Quantification of SIRT1-Dependent p53 Acetylation
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Briefly, 20 μg of cellular proteins were subjected to SDS-PAGE (NuPAGE precast gel, Thermo Fisher Scientific, USA) for electrophoresis and followed by dry transfer onto nitrocellulose membranes (iBlot Transfer Stack, Thermo Fisher Scientific, USA). The membranes were blocked with 5% BSA in TBS containing 0.1% (v/v) Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) for 60 minutes and incubated with the following primary antibodies: anti-human SIRT1 (1:2000 dilution), acetylated-p53 at K382 (Ac-p53; 1:500 dilution), AMPKα (1:4000 dilutuion), and p21 (1:2000 dilution) antibodies purchased from Cell Signaling Technology (Beverly, MA, USA), and p300 (1:2000 dilution) and total p53 (1:2000 dilution) from Santa Cruz (Dallas, USA). Anti-human GAPDH (1:5000 dilution), and β-actin (1:10000 dilution) antibodies were used as loading controls and purchased from Millipore (Billerica, USA) and Sigma Aldrich (St Louis, USA), respectively. Anti-rabbit and anti-mouse secondary antibodies (1:10000 dilutions) were bought from GE Healthcare (Buckinghamshire, United Kingdom). The immunoreactive bands were detected by an enhanced chemiluminescence system (Proteinsimple, Santa Clara, USA). Related signals were quantified using Proteinsimple image software (Santa Clara, USA).
3
IGF1 and Insulin Signaling Pathway
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Cells were treated with IGF1 or insulin for 24 h, after which they were washed with ice-cold phosphate-buffered saline (PBS) containing 5 mM EDTA and centrifuged at 1100 rpm. Lysis buffer was added and the cells were incubated on ice for 20 min. Cells were centrifuged at 13,000 rpm for 10 min and protein concentration was determined by the Bradford method. Samples were electrophoresed through 10% or 7.5% SDS-PAGE, followed by blotting of the proteins onto nitrocellulose membranes. After blocking with 5% skim milk, blots were incubated overnight with antibodies against OR5H2 (#102-12846, RayBiotech, Norcross, GA, USA), HSP70 (#7298, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and total p53 (mixture of #126 and #98, Santa Cruz Biotechnology). In addition, membranes were incubated with antibodies against IGF1R (#3027), total-ERK (#9102), phospho-ERK (#9101S), total-AKT (#9272), phospho-AKT (#9271S) and phospho-p53 (#9284). Antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibody, donkey anti-mouse and goat anti-rabbit were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
4
Protein Extraction and Immunoblotting Analysis
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Whole cell protein extracts were prepared by cell lysis with SDS-PAGE buffer (80 mM Tris-HCL pH 6.8, 16% glicerol, 4.5% SDS, 450 mM DTT, 0.01% bromophenol blue) with 200U/ml benzonase (Sigma) and 50 µM MgCl2 and boiling for 5 min. Equal amounts of protein extracts were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. Immunoblotting was performed with antibodies against the following proteins: H3K36me3 (ab9050; Abcam), histone H3 (ab1791; Abcam), phospho-53BP1 (phosphoSer1778; No.2675, Cell Signaling, Danvers, MA), phospho-ATM (phosphoS1981; No.200-301-400s, Rockland, Gilbertsville, PA), total ATM (PC116; Millipore), total RPA32 (ab2175; Abcam), phospho-RPA32 (phosphoSer4/Ser8; A300-245A, Bethyl, Montgomery, TX), α-Tubulin (T5168; Sigma); γH2AX (phosphoSer139; 05-636, Millipore); Histone H2B (ab1790; Abcam); GFP (11814460001; Roche, Basel, Switzerland); total p53 (sc-263; Santa Cruz); phospho-p53 (phosphoSer15; ab38497, Abcam); p21 (sc-397; Santa Cruz).
5
Western Blot Analysis of Heart Proteins
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The heart tissue was homogenated in extraction buffer (20 mmol/L Tris HCl, 1 mmol/L Na 3 VO 4 , 5 mmol/L NaF). The heart protein was collected and subjected to 10% or 15% SDS-polyacrylamide gel electrophoresis then transferred to polyvinylidene difluoride membranes, which were blocked for 2 h with 5% non-fat milk in Tris-buffered saline (pH 7.4) containing 0.1% Triton X-100. The membranes were probed overnight at 4 C with the appropriate primary antibody as follows: total-p38, phospho-p38, and phospho-HSP27 (Cell Signaling Technology, Danvers, MA), total-p53, phospho-p53, total-CREB, phospho-CREB, Bax, Bcl2, Cytochrome c, caspase-3 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA). After washing and exposure to horseradish peroxidaseconjugated secondary antibody for 1 h at room temperature, antibody-antigen complexes were visualized by enhanced chemiluminescence assay. Bands corresponding to the protein of interest appeared as dark regions on the developed film. The film images of the Western blots were scanned and were analyzed using Image J (NIH image, National Institutes of Health, Bethesda, MD) analysis software (Tanno et al., 2003) . For quantitation of the proteins of interest, phosphorylated proteins were normalized to total protein expression.
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