Figure 1: Biotin affinity purification method isolates efficiently the nuclear SMN complexes. (A) Schematic representation of DAP tag-fused SMN protein (HB-SMN-F). DAP-tag contains hexa-histidine sequence (6xHis) and biotinylated sequence (Biotin), whereas FLAG has a sequence of DYKDDDDK sequence. (B) 293TRex cells stably expressing HB-SMN-F-FLAG (HB-SMN-F cells) were fractionated into cytoplasmic extract (Cyto), cytoplasm wash fraction (Wash), and nuclear extract at low salt condition (Nuc: Low salt) or at high salt condition (Nuc: high salt) (see Materials and methods). They were analyzed by western blot method with the antibodies against FLAG and GAPDH, respectively. Parental 293TRex cells (293TRex cells) were used as a control. TCE; total cell extract. (C) SMN complexes were pulled down from the cytoplasmic (Cyto) or the nuclear (Nuc) extract of HB-SMN-F cells or 293TRex cells with anti-FLAG antibody (FLAG)- and Avidin (Biotin)-fixed beads, respectively, separated by SDS-PAGE, and visualized with silver staining. Parental 293TRex cells were used as a control. The proteins in the staining bands (1-4) are identified by LC-MS/MS-Mascot analysis, and the identified proteins are indicated at the right. (D) A MS/MS spectrum of a representative peptide assigned to the FOP (ASMQQQQQLASAR), expanding residues 39 (A) and 51 (R).