SBDS associates with nucleophosmin in an RNA-independent manner. (A) Endogenous SBDS was immunoprecipitated from HeLa nuclear extracts with an anti-SBDS antibody. The immunoprecipitated proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 4% to 12% Bis-Tris gel followed by Coomassie blue staining. Preimmune serum (Pre) was used as a negative control for the immunoprecipitation (lane 1). Protein bands from the SBDS immunoprecipitate (lane 2) were excised and analyzed by tryptic digestion and matrix-assisted laser description/ionisation time of flight (MALDI-TOF) mass spectrometry. (B) HeLa cell lysates were incubated with either anti-SBDS antibody or preimmune serum (Pre). The immunoprecipitates were analyzed by Western blotting for SBDS (lanes 1 and 2) and NPM (lanes 3 and 4). (C) 293T cells were transfected with either FLAG-NPM (lanes 2 and 4) or empty pFLAG-CMV vector (lanes 1 and 3). Cell lysates were immunoprecipitated with an anti-FLAG antibody. The resulting precipitates were immunoblotted for FLAG (lanes 1 and 2) or SBDS (lanes 3 and 4). (D) HeLa cell lysates were incubated at 30°C for 20 minutes in the presence (lane 2) or absence (lane 1) of 4 μg RNase A. RNA was extracted and analyzed by agarose gel electrophoresis and ethidium bromide staining (left panel, lanes 1 and 2). SBDS was immunoprecipitated from the mock-treated and RNase-treated lysates. The resulting pellets were analyzed by immunoblotting (right panel) for SBDS (lanes 3 and 4) and NPM (lanes 5 and 6).