Recombinant Human TP53

• Cat.No.: TP53-30262TH
• Product Overview: p53 Mutant Human Recombinant full length protein shows a 81 kDa band on SDS-PAGE.

Specification

• Species: Human
• Source: E.coli
• Tag: Non
• Description: This gene encodes tumor protein p53, which responds to diverse cellular stresses to regulate target genes that induce cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. p53 protein is expressed at low level in normal cells and at a high level in a variety of transformed cell lines, where its believed to contribute to transformation and malignancy. p53 is a DNA-binding protein containing transcription activation, DNA-binding, and oligomerization domains. It is postulated to bind to a p53-binding site and activate expression of downstream genes that inhibit growth and/or invasion, and thus function as a tumor suppressor. Mutants of p53 that frequently occur in a number of different human cancers fail to bind the consensus DNA binding site, and hence cause the loss of tumor suppressor activity. Alterations of this gene occur not only as somatic mutations in human malignancies, but also as germline mutations in some cancer-prone families with Li-Fraumeni syndrome. Multiple p53 variants due to alternative promoters and multiple alternative splicing have been found. These variants encode distinct isoforms, which can regulate p53 transcriptional activity.
• Form: Liquid
• Storage buffer: Preservative: NoneConstituents: 20% Glycerol, 50mM Tris acetate, 1mM EDTA, pH 7.5
• Storage: Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
• Full Length: Full L.

Gene Information

• Gene Name: TP53 tumor protein p53 [ Homo sapiens ]
• Official Symbol: TP53
• Synonyms: TP53; tumor protein p53; cellular tumor antigen p53; LFS1; Li Fraumeni syndrome; p53
• Gene ID: 7157
• mRNA Refseq: NM_000546
• Protein Refseq: NP_000537
• MIM: 191170
• Uniprot ID: P04637
• Chromosome Location: 17p13.1
• Pathway: Activation of BH3-only proteins, organism-specific biosystem; Activation of NOXA and translocation to mitochondria, organism-specific biosystem; Activation of PUMA and translocation to mitochondria, organism-specific biosystem; Amyotrophic lateral sclerosis (ALS), organism-specific biosystem; Amyotrophic lateral sclerosis (ALS), conserved biosystem
• Function: ATP binding; DNA binding; DNA strand annealing activity; MDM2 binding; RNA polymerase II transcription factor binding

Citation

c-Myc inactivation of p53 through the pan-cancer lncRNA MILIP drives cancer pathogenesis

Journal: Nature Communications    PubMed ID: 33020477    Data: 2020/10/5

Authors: Yu Chen Feng, Xiao Ying Liu, Lei Jin

Article Snippet:P53 protein concentrations were measured using PathScan Total p53 Sandwich ELISA Kit (Cell Signaling Technology, Cat#7370C), and Recombinant Human TP53 protein (Creative BioMart, Cat#TP53-15H) was used as a standard.. P53 protein copies per cell were calculated according to p53 concentrations and the cell equivalents of lysate.P53 protein copies per cell were calculated according to p53 concentrations and the cell equivalents of lysate.

a c-Myc bound to MILIP promoter in A549 and MCF-7 cells. An E-box motif, not associated with MYC target genes on Chr22, was used as a negative control. Data shown represent three independent experiments. ChIP, chromatin immunoprecipitation. b c-Myc silencing downregulated MILIP expression in A549 and MCF-7 cells. c-Myc responsive lncRNA OVAAL was used as a positive control. Data are mean ± s.d.; n = 3 independent experiments, one-way ANOVA followed by Tukey’s multiple comparisons test. c c-Myc silencing reduced the activity of reporters with intact c-Myc binding region (BR) of MILIP promoter but not that with the c-Myc-BR deleted (ΔBR) in A549 and MCF-7 cells. Data are mean ± s.d.; n = 3 independent experiments, two-tailed Student’s t test. d Representative microscopic photographs of in situ hybridization (ISH) analysis of MILIP expression in formalin-fixed paraffin-embedded (FFPE) lung adenocarcinoma (LUAD; n = 88 biologically independent samples) compared with paired adjacent normal tissues. Scale bar, 5 μm. e Quantitation of MILIP expression as detected in ( d ) in FFPE LUAD in comparison with paired adjacent normal tissues. RS, reactive score. Two-tailed Student’s t test. f Ingenuity Pathway Analysis (IPA) of RNA-seq data showing that p53 signaling was the most enriched pathway in A549 cells transfected with a MILIP siRNA (si-MILIP.2) relative to those introduced with the control siRNA. Orange bars represent pathways that were activated, and blue bars, inactivated. DEGs differentially expressed genes. g c-Myc knockdown upregulated p53 expression, which was diminished by MILIP overexpression in A549 and MCF-7 cells. Data are representatives or mean ± s.d.; n = 3 independent experiments, One-way ANOVA followed by Tukey’s multiple comparisons test. h c-Myc overexpression downregulated p53 expression, which was abolished by knockdown of MILIP in A549 and MCF-7 cells. Data shown represent three independent experiments. DOX, doxycycline, 200 ng/mL. Source data of Fig. 1a–c, e–h are provided as a Source Data file.

a Induced knockdown of MILIP by DOX reduced cancer cell anchorage-independent growth. Scale bar, 50 μm. Data are representatives or mean ± s.d.; n = 3 independent experiments, two-tailed Student’s t test. DOX: 200 ng/mL. b Induced knockdown of MILIP by DOX retarded A549 xenograft growth, which was reversed by DOX withdrawal in nu/nu mice. Data are mean ± s.d.; n = 6 mice per group, one-way ANOVA followed by Tukey’s multiple comparison test. DOX: 2 mg/mL supplemented with 10 mg/mL sucrose in drinking water. c MILIP silencing reduced A549 cell clonogenicity, which was attenuated by co-silencing of p53 siRNA1. Scale bar, 1 cm. Data are representatives or mean ± s.d.; n = 3 independent experiments, one-way ANOVA followed by Tukey’s multiple comparison test. d Kaplan–Meier analysis of the probability of overall survival (OS) of lung adenocarcinoma (LUAD; n = 477) and breast invasive carcinoma (BRCA; n = 1197) derived from the TCGA using the median/quartile of MILIP levels as the cut-off. e MILIP expression was increased in colon adenomas ( n = 15) compared with normal colon epithelia ( n = 16). Data are mean ± s.d.; two-tailed Student’s t test. f MILIP silencing decelerated anchorage-independent growth of HME-1 human mammary epithelial cells caused by c-Myc overexpression along with knockdown of p14 ARF . Scale bar, 50 μm. Data are representatives or mean ± s.d.; n = 2 independent experiments. g MILIP silencing decelerated anchorage-independent growth of MCF10A human mammary epithelial cells that did not express p14 ARF caused by c-Myc overexpression. Scale bar, 50 μm. Data are representatives or mean ± s.d.; n = 2 independent experiments. h MILIP overexpression caused anchorage-independent growth of MCF10A cells. Scale bar, 50 μm. Data are representatives or mean ± s.d.; n = 2 independent experiments. Source data of Fig. are provided as a Source Data file.

a Induced knockdown of MILIP prolonged the half-life time of p53 protein in cycloheximide (CHX)-chase assays. Data are representatives or mean ± s.d.; n = 3 independent experiments, two-tailed Student’s t test. CHX: 40 μg/mL, DOX: 200 ng/mL. b MILIP silencing reduced p53 polyubiquitination. Data shown represent three independent experiments. c Induced knockdown of MILIP did not reduce the binding between p53 and MDM2. Data shown represent three independent experiments. d MILIP was coprecipitated with p53 using RNA immunoprecipitation (RIP) assays. Data shown represent three independent experiments. e p53 was co-pulled down with MILIP by antisense probes against MILIP using RNA pulldown assays. Data shown represent three independent experiments. f In vitro transcribed MILIP was coprecipitated with purified Flag-tagged p53 in a cell-free system using RNA immunoprecipitation (RIP) assays. Data shown represent three independent experiments. g Overexpression of MILIP but not a MILIP mutant with the ?966/?1199 segment deleted (MILIP Δ?966/?1199) downregulated both endogenous (left) and overexpressed exogenous (right) p53 protein levels. Data are mean ± s.d.; n = 3 independent experiments, one-way ANOVA followed by Tukey’s multiple comparisons test. Source data of Fig. are provided as a Source Data file.