Journal: The Journal of Cell Biology
Article Title: Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization
doi: 10.1083/jcb.201601071
Figure Lengend Snippet: Fus-independent and dependent recruitment of paraspeckle proteins. (A) Simultaneous detection of Neat1 and seven of the protein components of paraspeckles, including Sfpq, Nono, Pspc1, Fus, Rbm14, Brg1, and Tardbp, in MEFs derived from WT and Fus KO mice. Note that DBHS family proteins (Sfpq, Nono, and Pspc1) and Tardbp, but not Rbm14 and Brg1, are recruited to the putative transcription site in the absence of Fus. Arrowheads indicate paraspeckle-like nuclear bodies formed at the putative Neat1 transcription site in Fus KO MEFs. (B) Simultaneous detection of various forms of NEAT1 and NONO in HAP1 cells and FUS-deleted HAP1 cells (ΔFUS HAP1). Probes used to detect NEAT1 are shown in the top boxes. (C) Schematic drawing of full-length and mutant FUS protein exogenously expressed by lentiviruses. ΔN FUS lack the PrLD and ΔC FUS lack the RNA binding domains including RNA recognition motifs (RRM) and arginine (R)-glycine-glycine domain (RGG) as well as zinc finger domain (ZF). (D) Western blot analyses of lysate from the cells infected with control EGFP (C), full-length FUS (FL), ΔN FUS (ΔN), and ΔC FUS (ΔC). Note that migration of FL and ΔN are much slower than predicted molecular mass (57 and 35 kD, respectively), probably because of the presence of PrLD in these molecules. (E) Simultaneous detection of Neat1 5′+3′ and Nono in Fus KO MEFs expressing various forms of FUS protein. Note that the core-shell structure of paraspeckles was rescued with FL FUS, but not with mutant molecules that lack either PrLD or RNA binding domains. (F) Confirmation of the specificity of polyclonal [Fus (poly)] and monoclonal (Fus) antibodies against Fus. Mixtures of MEFs derived from WT and KO mice of Fus were stained with each antibody. Note the complete absence of signals in the Fus KO MEFs (arrowheads). The positions of the epitope of these antibodies are shown in the schematic drawing of the domain structure of Fus. (G) Simultaneous detection of Fus using polyclonal antibodies and mAbs that recognize the N- and C-terminal region of the protein, respectively. Bars: (A, B, E, and G) 500 nm; (F) 200 µm.
Article Snippet: For the simultaneous detection of paraspeckle proteins, the following antibodies were used: mouse mAb against Sfpq (clone B92; Abcam), mouse mAb against Nono , mouse mAb against Pspc1 (clone 1L4; Sigma-Aldrich), mouse mAb against Fus (clone 4H11; Santa Cruz Biotechnology, Inc.), rabbit polyclonal antibody against Fus (ab84078; Abcam), rabbit polyclonal antibody against Brg1 (A300-813A; Bethyl Laboratories, Inc.), rabbit polyclonal antibody against Tardbp (10782-2-AP; Proteintech), and rabbit polyclonal antibody against Rbm14 (A300-311A; Bethyl Laboratories, Inc.).
Techniques: Derivative Assay, Mutagenesis, RNA Binding Assay, Western Blot, Infection, Control, Migration, Expressing, Staining
Bethyl is a verified supplier 
![Fus-independent and dependent recruitment of paraspeckle proteins. (A) Simultaneous detection of Neat1 and seven of the protein components of paraspeckles, including Sfpq, Nono, Pspc1, Fus, Rbm14, Brg1, and Tardbp, in MEFs derived from WT and Fus KO mice. Note that DBHS family proteins (Sfpq, Nono, and Pspc1) and Tardbp, but not Rbm14 and Brg1, are recruited to the putative transcription site in the absence of Fus. Arrowheads indicate paraspeckle-like nuclear bodies formed at the putative Neat1 transcription site in Fus KO MEFs. (B) Simultaneous detection of various forms of NEAT1 and NONO in HAP1 cells and FUS-deleted HAP1 cells (ΔFUS HAP1). Probes used to detect NEAT1 are shown in the top boxes. (C) Schematic drawing of full-length and mutant FUS protein exogenously expressed by lentiviruses. ΔN FUS lack the PrLD and ΔC FUS lack the RNA binding domains including RNA recognition motifs (RRM) and arginine (R)-glycine-glycine domain (RGG) as well as zinc finger domain (ZF). (D) Western blot analyses of lysate from the cells infected with control EGFP (C), full-length FUS (FL), ΔN FUS (ΔN), and ΔC FUS (ΔC). Note that migration of FL and ΔN are much slower than predicted molecular mass (57 and 35 kD, respectively), probably because of the presence of PrLD in these molecules. (E) Simultaneous detection of Neat1 5′+3′ and Nono in Fus KO MEFs expressing various forms of FUS protein. Note that the core-shell structure of paraspeckles was rescued with FL FUS, but not with mutant molecules that lack either PrLD or RNA binding domains. (F) Confirmation of the specificity of polyclonal [Fus (poly)] and monoclonal (Fus) antibodies against Fus. Mixtures of MEFs derived from WT and KO mice of Fus were stained with each antibody. Note the complete absence of signals in the Fus KO MEFs (arrowheads). The positions of the epitope of these antibodies are shown in the schematic drawing of the domain structure of Fus. (G) Simultaneous detection of Fus using polyclonal antibodies and mAbs that recognize the N- and C-terminal region of the protein, respectively. Bars: (A, B, E, and G) 500 nm; (F) 200 µm.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7409/pmc05037409/pmc05037409__JCB_201601071_Fig6.jpg)