1. Volume 2, Issue 4, Pages 799-806 (October 2012)
FUS-SMN Protein Interactions Link the Motor Neuron Diseases ALS and SMA 
Tomohiro Yamazaki, Shi Chen, Yong Yu, Biao Yan, Tyler C. Haertlein, Monica A. Carrasco, Juan C. Tapia, Bo Zhai, Rita Das, Melanie Lalancette-Hebert, Aarti Sharma, Siddharthan Chandran, Gareth Sullivan, Agnes Lumi Nishimura, Christopher E. Shaw, Steve P. Gygi, Neil A. Shneider, Tom Maniatis, Robin Reed 
Cell Reports 
Volume 2, Issue 4, Pages (October 2012)
DOI: /j.celrep
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2. Cell Reports 2012 2, 799-806DOI: (10.1016/j.celrep.2012.08.025)
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3. Figure 1 The SMN Complex and U1 snRNP Associate with FUS
(A) IP was carried out with HeLa nuclear extracts using FUS or negative control antibodies (lanes 1–3). In lane 3, nuclear extract was incubated with RNase A prior to IP. GST-FUS (lanes 4 and 5) or GST (lanes 6 and 7) was used for pulldowns from nuclear extract (lanes 4 and 6) or buffer alone (lanes 5 and 7). Proteins were run on a 4%–12% sodium dodecyl sulfate (SDS) gradient gel and detected by Coomassie staining. Indicated proteins were identified by MS. L, antibody light chain; ∗, FUS; ∗∗, a nonspecific band.
(B) Total RNA from IP and GST pulldown samples used in (A); 30% input (inpt) was loaded. RNAs were detected with ethidium bromide.
(C) Protein was isolated from samples in (B) followed by western blotting with the indicated antibodies; 15% input (inpt) was loaded.
(D) Table showing MS data for the indicated proteins from FUS IP. The number of total peptides (Total) and total unique peptides (Unique) identified by MS is shown.
(E) IP/western blotting with the indicated antibodies. The negative control for the FUS and TDP-43 antibodies was a rabbit polyclonal antibody (lane 2, SAP130), and the negative control for the monoclonal antibodies (SMN and Gemin3) was a monoclonal against HA (lane 7).
(F) Same as in (E) except that the nuclear extract was treated or not treated with RNase prior to the IP.
(G) FUS interacts directly with SMN. The indicated purified proteins (2 μg) were mixed in the presence of RNase A followed by GST pulldowns. Proteins were separated on a 4%–12% SDS-gradient gel and detected by Coomassie staining. Molecular weight markers in kDa are indicated.
See also Figure S1.
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4. Figure 2 FUS Is Required for Gem Formation in HeLa Cells
(A) FUS or control knockdown (KD) HeLa cells were used to detect Gems (green in nucleus) and FUS (red) by IF with SMN and FUS antibodies, respectively. FUS was knocked down using an shRNA against FUS. Scrambled shRNA was used as a negative control. DAPI shows the nucleus. Scale bar, 20 μm.
(B) IF staining of FUS or control KD HeLa cells was carried out with the indicated antibodies. Scale bar, 20 μm.
(C) Western analysis of FUS and control KDs in HeLa cells using the indicated antibodies. Tubulin was used as the loading control.
(D) SMN-GFP or GFP was expressed in control or FUS KD HeLa cells. Gems were detected with SMN-GFP and costaining with the Gemin3 antibody. DAPI shows the nucleus. Scale bar, 20 μm. Right panels (merged) show high magnification of the dashed squares indicated in the left panels.
See also Figure S2.
Cell Reports 2012 2, DOI: ( /j.celrep )
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5. Figure 3
Gems Are Lost in HeLa Cells Transfected with the ALS-Causing R495X FUS Mutation
(A) Representative images showing the expression of FLAG-tagged FUS or FUS R495X in HeLa cells (red). Gems were detected using SMN antibodies (green in nucleus). Scale bar, 20 μm.
(B) Quantitation of Gem levels in HeLa cells expressing the indicated proteins. The mean and standard deviation of Gem numbers per cell were calculated from three independent experiments. At least 100 cells were observed in each experiment. The p values were calculated by comparison with three controls (∗p < 0.01, Student’s t test).
See also Figure S3.
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6. Figure 4 Gems Are Deficient in FUS and TDP-43 ALS Patient Fibroblasts
(A) IF using the SMN antibody was used to detect Gems in fibroblasts from an unaffected individual and an ALS patient carrying a FUS R521C mutation. DAPI shows the nucleus. Scale bar, 20 μm.
(B) Same as in (A), except that fibroblasts bearing a TDP-43 M337V mutation were used.
(C) Graph showing Gem levels in ALS patients and unaffected individuals. The mean and standard deviation of Gem numbers per cell were calculated from three independent experiments. At least 150 cells were analyzed in each experiment. The p values were calculated by comparison with three controls (∗p < 0.01, Student’s t test). The age (years) and sex (M, male; F, female) of the individuals are indicated.
See also Figure S4.
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7. Figure S1
FUS and U1 snRNP Are Specifically Associated with FUS, and the RRM Motif of FUS Is Required for Binding U1 snRNP and the SMN Complex, Related to Figure 1
(A and B) Characterization of FUS rabbit polyclonal antibody. The FUS antibody detects one main band by western blots (A) and immunoprecipitates one main band (B). Note that FUS migrates at different positions on SDS versus SDS gradient gels. Molecular weight markers (in kDa) are indicated.
(C and D) The FUS antibody does not immunoprecipitate U1 snRNP in FUS knockdown nuclear extract. (C) U1-70K, U1A, SmB/B′, and SmD coimmunoprecipitate with FUS in sh control knockdown nuclear extract but not in sh FUS knockdown nuclear extract. (D) Same as in (C), but showing that U1 snRNA does not coimmunoprecipitate with FUS in FUS knockdown nuclear extract. Control antibodies in lanes 2 and 8 are mouse IgG, and those in lanes 6 and 12 are nonrelated rabbit serum.
(E) Schematic of FUS, showing domain organization. Schematics of truncated FUS proteins are shown below. Black shading indicates the FUS truncation containing the RRM domain, which is required for FUS binding to U1 snRNP and the SMN complex.
(F) Coomassie-stained gel (upper image) showing GST pulldowns from buffer (−) or nuclear extract (NE, +) using GST alone, full-length (FL) GST-FUS, or the indicated GST-FUS truncated proteins (∗ indicates FUS). Molecular weight markers in kDa are indicated. Western blots with the indicated antibodies using a portion of the samples are shown below.
(G) Ethidium bromide-stained gel showing total RNA isolated after GST pulldowns using the indicated FUS truncated proteins. The snRNAs and tRNA are indicated.
(H) Coomassie gel showing GST pulldowns from buffer (−) or nuclear extract (NE) (+) using GST alone, full length (FL) GST-FUS, or the indicated GST-FUS truncated proteins. ∗ indicates FUS. Molecular weight markers in kDa are indicated.
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8. Figure S2
SMN and Gemin3 Colocalize in Gems in HeLa Cells, Related to Figure 2
IF was performed with a rabbit polyclonal antibody against SMN and a mouse monoclonal antibody against Gemin3. DAPI was used to stain the nucleus. The merged images are shown.
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9. Figure S3
The NLS of FUS Is Not Required for Direct Interactions with SMN, Related to Figure 3
(A) Coomassie-stained gel showing GST pulldowns from buffer (−) or nuclear extract (NE) (+) using GST alone, full-length (FL) GST-FUS, or GST-FUS ΔNLS. Molecular weight markers in kDa are indicated.
(B) Western blots with the indicated antibodies using a portion of the samples in (A).
Cell Reports 2012 2, DOI: ( /j.celrep )
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10. Figure S4
Characterization of Human ALS Patient Fibroblasts and Analysis of Gem Levels in TDP-43 Knockdown HeLa Cells, Related to Figure 4
(A) Gems in normal human fibroblasts were detected by costaining with a mouse monoclonal (2B1) and rabbit polyclonal (H-195) antibody against SMN. DAPI and merged images are shown.
(B) FUS is mislocalized to the cytoplasm in ALS patient fibroblasts containing mutations in the FUS NLS. The indicated ALS patient or unaffected fibroblasts were stained with FUS antibodies.
(C) TDP-43 is localized in the nucleus in both normal and ALS patient fibroblasts containing TDP-43 mutations. The indicated ALS patient or unaffected fibroblasts were stained with TDP-43 antibodies.
(D) Knockdown of TDP-43 results in loss of Gems in HeLa cells. HeLa cells were transfected with siRNA against TDP-43 or nontargeting negative control siRNA. Cells were stained with TDP-43 or SMN, and DAPI was used to detect the nucleus. The TDP-43 staining shows that TDP-43 was efficiently knocked down, and the SMN staining shows that the level of Gems is significantly reduced.
Cell Reports 2012 2, DOI: ( /j.celrep )
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