Figure 2

Both TDP-43 and FUS/TLS bind to NEAT1_2 lncRNA and are colocalized with paraspeckle proteins. A. Characterization of NEAT1_2 foci and Cajal bodies (marker: coilin). After in situ hybridization using DIG-labeled NEAT1_2 probe, untransfected HeLa cells were triple-labeled with monoclonal IgG1 anti-DIG, monoclonal IgG2b anti-coilin and polyclonal anti-TDP-43 antibodies. Upper: NEAT1_2 lncRNA demonstrates a different localization pattern from Cajal bodies. Lower: endogenous TDP-43 overlaps with both NEAT1_2 foci and Cajal bodies. Dotted lines represent the outline of the nucleus. B. Using monoclonal antibodies, full-length TDP-43, FUS/TLS and β-actin bands are shown by immunoblotting. Immunoprecipitaion (IP) followed by solid washes in high-salt buffer purified the specific protein against each antibody. Protein-protein interactions were abolished. By combined RNAs, the complexes treated with 254 nm ultraviolet (UV) crosslinking and IP were shifted to the higher molecule compared to the bands of input without UV treatment. *Non-specific detections of the rabbit IgG heavy chains. C. NEAT1_2 lncRNA directly binds to TDP-43 and FUS/TLS. Following UV crosslinking in HeLa cells, NEAT1_2 RT-PCR bands are detected with the indicated number of PCR cycles after IP using each antibody. D. Immunofluorescence of HeLa cells, which exogenously expressed WT TDP-43 or FUS/TLS with the V5 tag, was carried out at 48 hours after transfection. Monoclonal or polyclonal anti-V5 along with anti-PSF or anti-PSP1 antibodies were used. Dotted lines represent the outline of the nucleus. Scale bars, 10 μm.