Phosphorylated cop1 molecules and uses thereof
A phosphorylation and molecular technology, applied in compound screening, analytical materials, biological material analysis, etc., can solve problems such as reducing p53
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preparation example Construction
[0166] Methods for preparing formulations known in the art can be found, for example, in "Remington's Pharmaceutical Sciences" (19th Edition), Ed. A. Gennaro, 1995, Mack Publishing Company, Easton, Pa. Formulations for parenteral administration may, for example, contain excipients, sterile water or saline, poly(alkylene) glycols such as polyethylene glycol, vegetable oils or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compounds. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients such as lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be in the form of nasal drops or Oily solution ...
Embodiment 1
[0172] Example 1: Materials and methods
[0173] Expression Vectors, Recombinant Proteins and Antibodies
[0174] FLAG-COP1, pcDNA3.1+p53, p21-Luc, bax-Luc, pGEX4T1-p53 and pGEX6P1-COP1 have been described previously 3，21 . The GST-S387 peptide contains amino acids 377-400 of the human COP1 polypeptide, while the GST-S387-A peptide has an alanine mutation at S387. FLAG-ATM and FLAG-ATM-KD were donated by Professor Michael Kastan (St. Jude Children’s Research Hospital, TN). All mutant constructs were generated by Quickchange site-directed mutagenesis (Stratagene). All GST recombinant proteins were expressed in E. coli BL21(DE3) codon+ (Stratagene) followed by purification using Glutathione Sepharose 4B batch method (GE Healthcare). Anti-p53(DO-1)(Calbiochem), Anti-p53 pS15(Cell Signaling), Anti-p21(Ab-1)(Calbiochem), Anti-FLAG(M2)(Sigma), Anti-Myc(9E10)(Roche), Anti-Muscle Actin (ICN), anti-tubulin (Ab6161) (Abcam), anti-histone H1 (SC8030) (Santa Cruz Biotechnology), an...
Embodiment 2
[0183] Example 2: COP1 is post-translationally modified upon exposure to IR
[0184] The present inventors examined COP1 steady state levels after induction of DNA damage with 10 Gy IR using GM0637 fibroblasts. Lysates probed with antibodies to COP1 revealed that steady-state levels of COP1 began to decline as early as 30 minutes after IR and nearly disappeared by 1 hour (Fig. 1A). Notably, as COP1 levels decreased, steady-state levels of p53 increased, consistent with activation of the downstream target gene p21 (Fig. 1A and 1B). To reveal the mechanism of this COP1 protein reduction, real-time PCR was performed to evaluate gene activity at the COP1 locus. COP1 mRNA levels were moderately elevated following IR stimulation. Two classical p53-dependent promoters, the p21 and PUMA promoters, were also induced after IR stimulation, although their magnitude was much larger than that of the COP1 promoter. Therefore, these data imply that decreased COP1 protein levels cannot be a...
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