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Fas associated factor 1

A technology related to factors and fragments, applied in the field of cell death, can solve problems such as unclear role

Inactive Publication Date: 2007-08-01
SK +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although hFAF1 appears to have multiple functions related to the apoptotic and ubiquitin pathways, its role in this regard is poorly understood

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 12

[0144] Example 1.2 - Cells and Reagents

[0145] at 37°C and 5% CO 2 Human embryonic kidney epithelial (HEK293T) cells were grown and maintained in high-glucose Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum. Monoclonal anti-Flag antibody M2 was purchased from Sigma. Monoclonal anti-Hsc70 / Hsp70 antibody was obtained from StressGen. Polyclonal anti-hFAF1 antibody was prepared by our research group.

[0146] Example 1.3 - Transient transfection

[0147] HEK293T cells were transfected with expression plasmids by calcium phosphate precipitation method. The day before transfection, cells were seeded on 10 cm dishes at a density of 1.5 × 10 6 cells were then transiently transfected with 6-12 μg of expression plasmid by means of calcium phosphate precipitation. The medium was replaced with fresh medium 6 hours after transfection, and cultured for an additional 18 hours.

Embodiment 14

[0148] Example 1.4 - Metabolic labeling and immunoprecipitation

[0149] Use 2 μCi / ml in methionine semi-free medium [ 35 S] Methionine metabolically labels cells for 18 hours. On ice with buffer containing protease inhibitors (50mM Tris, pH8.0, 150mM NaCl, 2mM EDTA, 0.5% NP-40, 1mM PMSF, 0.5mMDTT, 5μg / ml aprotinin, 1μg / ml leupeptin Peptide, 5mM Na 3 VO 4 , 5mMNaF) to disrupt the cells for 30 minutes. Centrifuge the lysate at 12,000 rpm for 1 h, then incubate the supernatant with monoclonal anti-Flag M2-affinity cross-linked agarose beads for 3 h at 4 °C, or incubate with anti-Flag antibody for 2 h and at 4 °C. Incubate with protein A beads for an additional 1 hour. The beads were washed three more times with 1 ml of lysis buffer.

[0150] Example 1.5 - Two-dimensional gel electrophoresis

[0151] At room temperature with buffer (containing 9.5M urea, 2% Triton X-100, 5% β-mercaptoethanol, 1mM PMSF, 5μg / ml aprotinin, 10ug / ml pepstatin A, 10μg / ml light Aprotinin, 1 ...

Embodiment 17

[0154] Example 1.7 - Confocal Microscopy

[0155]For morphological studies, cells were grown on coated coverslips, transiently transfected, and then treated with heat shock. The cells were rinsed gently in HBSS and then fixed with 4% paraformaldehyde in HBSS for 10 minutes at room temperature. After washing with HBSS, cells were permeabilized by incubation with 0.1% Triton X-100 in HBSS for 10 min at room temperature before immunostaining. Nonspecific protein uptake was inhibited by incubating cells in HBSS (containing 3% BSA, 0.2% Tween 20, and 0.2% gelatin) for 1 hour. For Hsc70 / Hsp70 staining, cells were incubated with mouse monoclonal Hsc70 / Hsp70 antibody (StressGen) diluted 1:150 in HBSS (with 1% sucrose and 1% BSA) for 2 hours at 37°C. After washing the wash three times with HBSS, cells were incubated with Texas red-conjugated rabbit anti-mouse (Molecular Probes) diluted 1:200 for 1 hour, followed by washing with HBSS. Flag-hFAF1 was stained with FITC-labeled monocl...

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Abstract

The present application describes a Fas associated Factor 1 protein and fragment thereof that bind Hsc70 / Hsp70, ubiquitinated protein and valosin containing protein.

Description

technical field [0001] The present invention relates to Fas associated factor 1 (Fas associated factor 1, FAF1) and its fragments. The invention also relates to the field of cell death. The present invention further relates to inhibiting the chaperone activity of HSP70 and inhibiting the degradation of ubiquinated protein. The invention further relates to methods for screening for cell death inducing molecules. The present invention also relates to cancer diagnostic markers. The invention also relates to cancer treatment. The invention further relates to the treatment of (cellular) hyperplasia or rapid cell growth. In addition, the present invention relates to the treatment of diseases caused by cell death by inhibiting FAF1 activity. Background technique [0002] Fas-associated factor 1 (FAF1) was first identified as a binding protein for the cytoplasmic region of Fas in a mouse yeast two-hybrid screen. Transient overexpression of mouse FAF1 (mFAF1 ) enhances Fas-indu...

Claims

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Application Information

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IPC IPC(8): C07K14/47A61K38/17A61P35/00A61P43/00G01N33/574
Inventor 李公珠金禧廷赵政宇金银姬宋垠妵孟哲永
Owner SK
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