Figure 3: FOP-associating nuclear SMN complex lacks Gemin5 and coilin as well as U1 and U2 snRNAs. (A) Schematic representation of FOP tagged with DAP lacking FLAG (HB-FOP). (B) 293TRex cells stably expressing HB-FOP (HB-FOP 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 Material and methods). They were analyzed by western blot method with the antibodies against Lamin B and GAPDH, respectively. HB-FOP was detected with HRP-conjugated streptavidin. Parental 293TRex cells were used as a control (293TRex cells). (C) HB-FOP complex was pulled down from the nuclear extract of HB-FOP cells or 293TRex cells with avidin-fixed beads (Biotin), separated by SDS-PAGE and visualized by silver staining. The protein band of HB-FOP was assigned based on its mobility expected from the amino acid sequence of HB-FOP on SDS-PAGE gel. Molecular weights of the marker proteins are indicated at left. (D) The pulled-down HB-FOP complex were analyzed by western blot with the antibodies indicated at the right. HB-FOP was detected with HRP-conjugated streptavidin. Total RNA (1 μg) was used as input. (E) The RNAs extracted from the HB-FOP complex were analyzed by northern blot with the biotin labeled DNA probes complementary to U1-U6 sn(o)RNAs, respectively. (F) The pulled-down HBFOP complex was analyzed by western blotting with the antibodies against proteins indicated at the right with (+) or without (-) RNase A treatment. U6 snRNA was detected by northern blot analysis. (G) 293TRex cells inducibly expressing FLAG-fused-FOP (FLAG-FOP cells) were subjected to immunocytochemical analysis with the antibody against FLAG (green) or endogenous SMN (red) after the removal of the cytoplasmic proteins by permeabilization with Triton. DAPI staining (blue) shows the nucleus. Scale bar: 10 μm.