RNA immunoprecipitation

H9 cells grown on Matrigel were UV-cross-linked at 0.15 mJ/cm² (254 nm). Cells were harvested and snap-frozen. The cell pellets were resuspended in lysis buffer (50 mM Tris–HCl at pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, with protease inhibitors), and the DAP5–RNA complexes were immunoprecipitated with rabbit anti-DAP5 monoclonal antibody (Cell Signaling, 5169) followed by stringent washes with up to 1 M NaCl. The bound RNA was extracted with TRI reagent, cDNA was generated, and quantitative RT–PCR was performed as described. RNA immunoprecipitation with anti-DAP5 antibody was normalized to the corresponding levels within total mRNA (input RNA).