mouse anti human phospho p53  (Cell Signaling Technology Inc)

Journal: Mechanisms of ageing and development

Article Title: Cellular senescence in vascular wall mesenchymal stromal cells, a possible contribution to the development of aortic aneurysm.

doi: 10.1016/j.mad.2021.111515

Figure Lengend Snippet: Fig. 5. (A and B) representative images of immunofluorescence labeling of p – H2AX in h – MSCs and AAA – MSCs isolated from two different donors. A FITC conjugated secondary antibody was used to detect the nuclear localization of p – H2AX. AAA – MSCs nuclei showed a stronger green fluorescent signal compared to h – MSCs nuclei (magnification 600X; bar: 100 nm); (C) representative western blot images showing total p53 and P-p53 protein expression in h – MSCs and AAA – MSCs. Results from two different donors were shown. (D) Relative amounts of total p53 and P-p53 protein expression were normalized to the intensity of actin and represented as fold increase relative to h – MSCs of each donor. Western blotting was performed in duplicate and the relative quantification was expressed as mean value ± SD. * represents a significant difference compared to h - MSCs, p < 0.05 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Article Snippet: The membranes were blocked with dry milk (blocking reagent) (Invitrogen, Thermo Fisher Scientific, Monza, Italy) for 30 min at room temperature and were then incubated with the following primary antibodies: mouse anti-human p53 antibody (Cell Signaling Technologies, Euroclone, Milan, Pero); mouse anti-human phospho - p53 (Ser 15) (Cell Signaling Technologies, Euroclone, Milan, Italy); rabbit anti-human p21CIP antibody (Cell Signaling Technologies, Euroclone, Milan, Italy); rabbit anti human p16INK4A antibody (Cell Signaling Technologies, Euroclone, Milan, Italy); rabbit anti-human Beclin (Cell Signaling Technologies, Euroclone, Milan, Italy); rabbit anti-human LC3 (Cell Signaling Technologies, Euroclone, Milan, Italy); mouse anti-human tubulin antibody (Sigma- Aldrich, St Louis, Missouri, USA) and mouse anti-human actin antibody (Millipore Merck, Darmstadt, Germany).

Techniques: Immunofluorescence, Labeling, Isolation, Western Blot, Expressing, Quantitative Proteomics

Journal: Cell Reports

Article Title: p53 Loss in Breast Cancer Leads to Myc Activation, Increased Cell Plasticity, and Expression of a Mitotic Signature with Prognostic Value

doi: 10.1016/j.celrep.2018.12.071

Figure Lengend Snippet: Myc is Overexpressed in Murine and Human Breast Tumors Because of Attenuation or Loss of p53 Signaling (A and B) Western blot of Myc protein expression in (A) WT mammary gland and 5 independent ErbB2 tumors (T1–T5), and (B) two representative ErbB2 tumor mammosphere cultures (T19 and T20), untreated or treated with Nut3 (2.5 μM), as compared with one WT mammosphere culture. Mammospheres were analyzed at passage 3 (M3). Right: Relative Myc-protein expression for the untreated and Nut3-treated T19 and T20. (C) Representative FACS-histograms of EdU (5-ethynyl-2′-deoxyuridine) incorporation in WT and ErbB2-tumor (T) cells at different times during mammosphere formation, as indicated. The percentage of EdU + cells (cells in S phase) is shown for each time point. (D and E) Western blot of Myc expression: (D) during WT and ErbB2 tumor mammosphere formation (24–120 h); and (E) in MCF10DCIS.com and primary cells from four PDX tumors (BC3, BC10, BC22, and BC26), untreated (UT) or treated with 2.5 or 10 μM Nut3 for 16 h in vitro . Right: Relative Myc-protein expression. See also Figure S1 .

Article Snippet: Rabbit polyclonal anti-human/mouse phosphoS15 p53 , Cell Signaling , Cat#9284; RRID: AB_331464.

Techniques: Western Blot, Expressing, In Vitro

Journal: Cell Reports

Article Title: p53 Loss in Breast Cancer Leads to Myc Activation, Increased Cell Plasticity, and Expression of a Mitotic Signature with Prognostic Value

doi: 10.1016/j.celrep.2018.12.071

Figure Lengend Snippet: Myc Is the Downstream Effector of p53 Loss in Breast CSCs (A) Relative sphere number of WT (n = 2) and ErbB2-tumor (ErbB2; n = 3) cells transduced with the TET-inducible Omomyc vector. Spheres were counted at the end of the second passage in the absence (UT) or presence of 0.5 μM Doxycycline (+Dox). ∗ paired t test p < 0.001. (B and C) Cumulative sphere (B) and cell (C) number graphs of LTR-Ctrl and LTR-MycER ErbB2-tumor mammospheres (n = 4), in the absence or presence of 2.5 μM Nut3. ∗ paired t test p < 0.05. (D) Top: schematic representation of the experimental outline. Mice transplanted with LTR-Ctrl (n = 13) or LTR-MycER (n = 12) ErbB2-tumor cells were intraperitoneally [i.p.] injected with vehicle (DMSO, n = 7 for LTR-Ctrl and n = 6 for LTR-MycER) or with Nut3 (n = 6 for both LTR-Ctrl and LTR-MycER). Bottom: box plot of tumor volumes for each experimental group at the end of treatment. ∗ t test p < 0.05. (E) Tumor-free survival curve for mice injected with LTR-Ctrl and LTR-MycER DCIS cells untreated or Nut3-treated as mammospheres in vitro . Numbers of mice per group as indicated. ∗ Mantel-Cox test between LTR-Ctrl treated versus untreated, p < 0.01. (A–C) Error bars represent SD. See also Figure S5 .

Article Snippet: Rabbit polyclonal anti-human/mouse phosphoS15 p53 , Cell Signaling , Cat#9284; RRID: AB_331464.

Techniques: Transduction, Plasmid Preparation, Injection, In Vitro

Journal: Cell Reports

Article Title: p53 Loss in Breast Cancer Leads to Myc Activation, Increased Cell Plasticity, and Expression of a Mitotic Signature with Prognostic Value

doi: 10.1016/j.celrep.2018.12.071

Figure Lengend Snippet: p53 and Myc Co-regulate Mitotic Genes in CSCs (A) Genome browser visualization of p53 binding on the c-myc gene. (B) GSEA pre-ranked analysis showing enrichment of the Myc signature in the p53 −/− . Left: upregulated genes; Right: downregulated genes. NES, normalized enrichment scored. (C) Venn diagram of p53-Myc DEGs in non-transformed mammospheres: intercross between p53-DEGs and Myc-DEGs. Numbers represent genes coherently up- and downregulated and relative percentages. (D) GSEA pre-ranked analysis, as in (B) showing enrichment of the Myc signature in the ErbB2-DEGs (top) and the Nut3-DEGs (bottom). (E) Left, Venn diagram of p53 and/or Myc DEGs in ErbB2 tumors: intercross between the union of p53- and Myc-DEGs (red circle) and ErbB2-DEGs (blue circle). Right: pie chart, characterization of the p53 and/or Myc DEGs in only p53-dependent (blue), only Myc-dependent (red), and common p53-Myc DEGs (green). (F) Pathway analysis of the ErbB2 p53-Myc common DEGs using MSigDB ( http://software.broadinstitute.org/gsea/msigdb/index.jsp ). The top 15 up- and downregulated pathways are shown ranked by q-value. See also Figure S6 and and .

Article Snippet: Rabbit polyclonal anti-human/mouse phosphoS15 p53 , Cell Signaling , Cat#9284; RRID: AB_331464.

Techniques: Binding Assay, Transformation Assay, Software

Journal: Cell Reports

Article Title: p53 Loss in Breast Cancer Leads to Myc Activation, Increased Cell Plasticity, and Expression of a Mitotic Signature with Prognostic Value

doi: 10.1016/j.celrep.2018.12.071

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-human/mouse phosphoS15 p53 , Cell Signaling , Cat#9284; RRID: AB_331464.

Techniques: Derivative Assay, Drug discovery, Biomarker Discovery, Recombinant, Flow Cytometry, Membrane, Labeling, Gene Expression, Microarray, Software

Journal: PLoS ONE

Article Title: Sterigmatocystin-Induced DNA Damage Triggers G 2 Arrest via an ATM/p53-Related Pathway in Human Gastric Epithelium GES-1 Cells In Vitro

doi: 10.1371/journal.pone.0065044

Figure Lengend Snippet: GES-1 cells were treated with different concentrations of ST (0.075, 0.3, 1.5, and 3 µM) or solvent for 48 h. (A) Representative immunoblots show the effect of ST treatment on the phosphorylation of p53 (Ser-15) and the expression of p53 and p21. β-actin was used as the normalization control. (B) Intensities of the immunoreactive bands were quantified by densitometric scanning and compared with those of the control (considered “1”). The values shown represent the means ± SD. * P <0.05 compared with the solvent-treated control group.

Article Snippet: The primary antibodies used for the Western blot analysis were mouse anti-human Cyclin B1 antibody (eBioscience, CA, USA), rabbit anti-human Cdc2, Cdc25C, ATM, phospho-ATM (Ser-1981), and phospho-Chk2 (Thr-68) monoclonal antibodies (Epitomics, CA, USA), rabbit anti-human Chk2 monoclonal antibodies (Millipore, MA, USA), rabbit anti-human phospho-Cdc2 (Tyr15) and phospho-Cdc25C (Ser216) monoclonal antibodies (Cell Signaling Technology, MA, USA), mouse anti-human phospho-p53 (Ser15) monoclonal antibody (Cell Signaling Technology, MA, USA), rabbit anti-human p53, Bax, and caspase-3 antibodies, and mouse anti-human p21 waf1 and Bcl-2 antibodies (Santa Cruz, CA, USA).

Techniques: Solvent, Western Blot, Phospho-proteomics, Expressing, Control

Journal: PLoS ONE

Article Title: Sterigmatocystin-Induced DNA Damage Triggers G 2 Arrest via an ATM/p53-Related Pathway in Human Gastric Epithelium GES-1 Cells In Vitro

doi: 10.1371/journal.pone.0065044

Figure Lengend Snippet: Cells were treated with the indicated agent for 48 h (pretreatment with 5mM caffeine for 2 hours followed by ST treatment). (A) Caffeine blocked the phosphorylation of ATM (Ser-1981), Chk2 (Thr-68), and p53 (Ser-15) and downregulated the expression of p21 stimulated by ST exposure. (C) Caffeine affected the G 2 /M phase regulatory proteins that were altered by ST treatment. β-actin was used as the loading control. (B, D) Intensities of the immunoreactive bands in “A” and “C” were quantified by densitometric scanning and compared to those of the control (considered “1”). (E) Caffeine effectively prevented the G 2 arrest induced by ST, as demonstrated by flow cytometric analysis. The data represent the means ± SD of three independent determinations. * P <0.05, compared with the solvent-treated control group. ▴ P <0.05 compared with the ST-treated group.

Article Snippet: The primary antibodies used for the Western blot analysis were mouse anti-human Cyclin B1 antibody (eBioscience, CA, USA), rabbit anti-human Cdc2, Cdc25C, ATM, phospho-ATM (Ser-1981), and phospho-Chk2 (Thr-68) monoclonal antibodies (Epitomics, CA, USA), rabbit anti-human Chk2 monoclonal antibodies (Millipore, MA, USA), rabbit anti-human phospho-Cdc2 (Tyr15) and phospho-Cdc25C (Ser216) monoclonal antibodies (Cell Signaling Technology, MA, USA), mouse anti-human phospho-p53 (Ser15) monoclonal antibody (Cell Signaling Technology, MA, USA), rabbit anti-human p53, Bax, and caspase-3 antibodies, and mouse anti-human p21 waf1 and Bcl-2 antibodies (Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Expressing, Control, Solvent

Journal: PLoS ONE

Article Title: Sterigmatocystin-Induced DNA Damage Triggers G 2 Arrest via an ATM/p53-Related Pathway in Human Gastric Epithelium GES-1 Cells In Vitro

doi: 10.1371/journal.pone.0065044

Figure Lengend Snippet: Cells were either not transfected or transfected with 100 nM p53 siRNA and then treated with 3 µM ST for 48 h. (A) Cells were subjected to immunoblot analysis for p-p53 (Ser15), p53, p21, and (C) the regulators related to G 2 arrest. NC: cells transfected with the same concentration of negative control siRNA. β-actin was used as the loading control. (B, D) Intensities of the immunoreactive bands in “A” and “C” were quantified by densitometric scanning and compared with those of the control (considered “1”). (E) The cell cycle phases of the cells were analyzed by FCM. The values shown represent the means ± SD, * P <0.05 compared with the solvent-treated control group. ▴ P <0.05 compared with the ST-treated groups. # P <0.05 compared with the p53 siRNA-treated groups.

Article Snippet: The primary antibodies used for the Western blot analysis were mouse anti-human Cyclin B1 antibody (eBioscience, CA, USA), rabbit anti-human Cdc2, Cdc25C, ATM, phospho-ATM (Ser-1981), and phospho-Chk2 (Thr-68) monoclonal antibodies (Epitomics, CA, USA), rabbit anti-human Chk2 monoclonal antibodies (Millipore, MA, USA), rabbit anti-human phospho-Cdc2 (Tyr15) and phospho-Cdc25C (Ser216) monoclonal antibodies (Cell Signaling Technology, MA, USA), mouse anti-human phospho-p53 (Ser15) monoclonal antibody (Cell Signaling Technology, MA, USA), rabbit anti-human p53, Bax, and caspase-3 antibodies, and mouse anti-human p21 waf1 and Bcl-2 antibodies (Santa Cruz, CA, USA).

Techniques: Transfection, Western Blot, Concentration Assay, Negative Control, Control, Solvent

Journal: PLoS ONE

Article Title: Sterigmatocystin-Induced DNA Damage Triggers G 2 Arrest via an ATM/p53-Related Pathway in Human Gastric Epithelium GES-1 Cells In Vitro

doi: 10.1371/journal.pone.0065044

Figure Lengend Snippet: In response to ST-induced DNA damage, ATM serves as a signal transducer for the activation of its downstream signaling pathway. Activated ATM simultaneously phosphorylates the Thr-68 and Ser-15 residues of Chk2 and p53, respectively. These phosphorylations lead to the activation of their downstream pathway components, which results in the inhibition of the activation of Cdc25 and an increase in the expression of p21 waf1 . These steps finally result in the inactivation of the Cyclin B1/Cdc2 complex and the induction of G 2 arrest.

Article Snippet: The primary antibodies used for the Western blot analysis were mouse anti-human Cyclin B1 antibody (eBioscience, CA, USA), rabbit anti-human Cdc2, Cdc25C, ATM, phospho-ATM (Ser-1981), and phospho-Chk2 (Thr-68) monoclonal antibodies (Epitomics, CA, USA), rabbit anti-human Chk2 monoclonal antibodies (Millipore, MA, USA), rabbit anti-human phospho-Cdc2 (Tyr15) and phospho-Cdc25C (Ser216) monoclonal antibodies (Cell Signaling Technology, MA, USA), mouse anti-human phospho-p53 (Ser15) monoclonal antibody (Cell Signaling Technology, MA, USA), rabbit anti-human p53, Bax, and caspase-3 antibodies, and mouse anti-human p21 waf1 and Bcl-2 antibodies (Santa Cruz, CA, USA).

Techniques: Activation Assay, Inhibition, Expressing