YK 4-279, an inhibitor of RNA Helicase A (RHA), binds to the oncogenic transciption factor EWS-FLI1.

Cytosolic phosphorylation of Akt: Hela cells are serum starved for 1 hr and treated with IGF (100ng/mL) or SC79 (4 μg/mL) for 30 minutes. Cells are lysed in Lysis buffer containing 250 mM Sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA supplemented with protease inhibitors. Cells are passed through 25 g needle several times and kept on ice for 20 minutes. Total cell lysate is taken at this point. Cell lysates are centrifuged at 100,000 g for 30 minutes. Supernatant is collected as the cytosolic fraction. Pellet is washed with lysis buffer and represents the membrane fraction. Total cell lysate, cytosolic and membrane fractions are resolved by SDS-PAGE and analyzed for phospho-Akt (S473), Total Akt, Tubulin (cytosolic marker) and Orai1 (membrane marker) by western blotting.