Gel electrophoresis profile of the TRBP-interacting proteins. (A) Total cell lysates from HeLa cells overexpressing Flag-TRBP were collected and immunoprecipitated with monoclonal anti-Flag agarose. Flag-TRBP and its interacting proteins were eluted with 3X Flag polypeptides and were subjected to sodium dodecyl-sulfate (SDS) gel electrophoresis followed by colloidal Coomassie staining. Co-immunoprecipitated products using cell lysates with mock transfection were compared. Fifteen gel bands as marked from the mock and TRBP eluents were excised for mass spectrometry analyses. (B) The immunoprecipitated Flag-agarose beads were treated with RNase A for 1 hour and washed three times with RIPA buffer. The remaining binding proteins were eluted with 3X Flag polypeptides and analyzed by gel-electrophoresis and silver staining. Please see Additional file 2, supplementary Materials and Methods, for detailed methodology.