2.8. huRNP U Prevents RPA2 from Binding Telomeres

E. coli-derived Flag-hnRNP U 683C was immunoprecipitated with anti-Flag M2-agarose affinity gel, as described above. After the removal of E. coli-derived DNA, complexes were mixed with 20 µg of mouse embryonic stem cell extract and whole cell extract (WCE) and 10 µL of 10 µM ssTel or ssTel(da) for 3 h at 4 °C. After centrifugation at 2000 rpm for 5 min at 4 °C, supernatants were transferred into a new tube and mixed with 20 µL streptavidin-DynaBeads and mixed for 1 h at 4 °C and washed three times with KCl buffer. Resulting pellets were subjected to Western blotting with anti-RPA2 antibody, as described above.