Association of SBDS point mutants with the 60S ribosomal subunit. (A) Marrow stromal cells from SDS patients were infected with FLAG-tagged wild-type SBDS (WT) or SBDS N-terminal deletion mutants lacking the first 5, 10, or 15 amino acids (del 5 AA to del 15 AA). Protein expression was determined with an anti-FLAG Western blot of whole-cell lysates. (B) Cells harboring the indicated SBDS cDNAs were lysed in 0.25mM MgCl2, followed by sucrose density fractionation. Proteins were precipitated from each gradient fraction and immunoblotted with an anti-FLAG antibody (bottom panel). (C) A Coomassie stain of the indicated purified recombinant SBDS proteins is shown. (D) Equimolar quantities of recombinant SBDS proteins were added to SDS lymphoblast lysates prepared in 0.25mM MgCl2 to dissociate the ribosomal subunits. Lysates were fractionated by sucrose density sedimentation to identify 40S and 60S subunit peaks. Proteins were precipitated from equal aliquots of each gradient fraction and immunoblotted for SBDS.
