Figure 4: SILAC mass spectrometry analysis of the nuclear SMN complexes. 293EBNA cells were cultured in heavy medium containing 13C lysine and 13C,15N arginine and transfected with siRNA for knockdown of FOP (si), whereas those in light medium containing 12C lysine and 12C,14N arginine were transfected with scRNA (sc). (A) The efficiency of the knockdown was examined by western blot analysis with the antibody against GAPDH and FOP. The nuclear SMN complexes immunoprecipitated with anti-SMN antibody were separated by SDS-PAGE and stained with colloidal blue. The resulting gel was cut into 16 pieces and each of the gel pieces was analyzed by the LC-MS/MS analysis after in gel trypsin digestion. (B) Typical MS/MS survey scans from a LTQ-Orbitrap mass spectrometer. (Upper left) Mass region containing heavy (labeled with 13C6, 15N4-Arg and 13C6-Lys) and light (labeled with 12C6,14N4-Arg and 12C6-Lys) forms of GTGQSDDSDIWDDTALIK from the SMN protein (SMN) is shown. Heavy/Light=1.02. (Upper right) The region containing heavy and light forms of VFLENVIR from the Histone H4 (H4). Heavy/Light=0.324. (Bottom left) The region containing heavy and light forms of LFIGGLNVQTSESGLR from the heterogeneous nuclear ribonucleoprotein A0 protein (ROA0). Heavy/ Light=0.378. (Bottom right) The region containing heavy and light forms of LFVGNLPADITEDEFK from the Splicing factor, proline- and glutamine-rich protein (SFPQ). Heavy/Light=0.561. (C) Heavy/Light ratios of the components of the endogenous nuclear SMN complexes quantified by SILAC-LC-MS/MS method are given. Vertical axis; Heavy/Light ratio, Horizontal axis; proteins identified. Error bar is given in each bar. Open bars show proteins with the ratios over 1.5 or below 0.66. Striped bars show the components of the nuclear SMN complexes.