Background
Methods
RNA Extraction and cDNA preparation
In-silico analysis of EWS-FLI1 protein using prime and SiteMap
Cloning and expression of EWS-FLI1
Immunoblotting
Purification of recombinant EWS-FLI1
Tryptic mapping and mass spectrometry
Fold index plot
Purification of soluble junction construct
Dialysis and circular dichroism
Expression of short junction region (aa 251 to aa 280) in Ewing Sarcoma cells
Colony forming assay
Real-Time quantitative PCR
Results
Junction region (aa 251 to aa 343) encompasses potential ligand binding sites in the EWS-FLI1 protein
SITE | Residue Number | Score |
---|---|---|
4 | 21,156–167,170,173,176-196,203-207,218- 223 247,248, 317–327, 389, 391, 394,447-450,454,498 | 1.151 |
3 | 109-112,117,120,121,124,125,128,129,146-148,150-153,298 305, 307, 308,310,315,317,324,325,327,328,331,332,335,345,347,380,382,383,385-389, 481-490 | 1.077 |
2 | 12-16,54-64,66-69,81-86,89-98,100-103,105,107,109,112,119 – 124, 127 , 128,132-146,272,276,290,296,311 | 1.047 |
6 | 25,65,71-75,77,209,213-217,236-247,250,254-264,278-286
| 1.043 |
5 | 1,14-16,29,31,32,35,36,41-44,49,50,54-59,66,67,90,92-97,261-264,267-272
| 1.032 |
9 | 150,151,158-160,315,317-329,350,377-380,387-391,456,461,489-492 | 1.026 |
1 | 22,25-34,36-40,163,188,190-211,214,216-218,246-254,257-259, 261,262,264,279-282,318,352,365-369,377,378,391-401,436,439-450,454 | 1.007 |
8 | 179-181,184,206-208,216,219-223,229-237,240,244,246,285,288,289
| 0.944 |
7 | 11,104,106,144-147,307-314,374,383-388,410-413,476,479-482,484 | 0.948 |
10 | 61-63,80-86,89,118,119,223,234,235,288-296 | 0.777 |