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. 2018 Aug 27;8:12901. doi: 10.1038/s41598-018-31078-1

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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The RNA exosome and the NEXT complex interact with the splicing factor SRSF3. (a) HA-SRSF3 was co-expressed with individual Flag epitope-tagged RNA exosome subunits in HeLa cells as indicated. The top panels show western blotting of 1/10 of total extract (‘Input’), and the bottom panels show western blotting following immunoprecipitation with an anti-Flag antibody (IP: Flag). Anti-hRRP6 antibody was used as a positive control. (b) and (c) HA-SRSF3 and Flag-hMTR3 were expressed by transient transfection in HeLa cells as indicated. Cellular extracts were immunoprecipitated with an anti-Flag antibody (b) or an anti-HA antibody (c) in the presence or absence of RNase A. The immunoprecipitated complexes were then analyzed by western blotting using an anti-Flag polyclonal antibody (c) or an anti-HA antibody (b). (d) and (e) HA-SRSF3 was coexpressed with individual Flag epitope-tagged hTRAMP (d) or NEXT (e) complex subunits in HeLa cells. Cellular extracts were immunoprecipitated with an anti-Flag antibody and then the immunoprecipitated complexes were analyzed by western blotting using an anti-Flag polyclonal antibody to visualize the hTRAMP and NEXT complex subunits (top panel), or an anti-HA antibody to detect SRSF3 (bottom panel). (f) and (g) HA-SRSF3 and RBM7 Flag-tagged NEXT subunit were expressed by transient transfection in HeLa cells as indicated. Cellular extracts were immunoprecipitated with an anti-Flag antibody (f) or with an anti-HA antibody (g) in the presence or absence of RNase A. The immunoprecipitated complexes were then analyzed by western blotting using an anti-Flag polyclonal antibody (g) or an anti-HA antibody (f). (h) HA-SRSF3 was expressed by transient transfection in HeLa cells previously treated with control or specific siRNAs for the NEXT complex, either alone or together with the hMTR3 Flag-tagged RNA exosome subunit. Cellular extracts were immunoprecipitated with an anti-Flag antibody in the presence or absence of RNase A. The immunoprecipitated complexes were then analyzed by western blotting using an anti-Flag polyclonal antibody to visualize hMTR3 (top panel), anti-RBM7 or anti-ZCCHC8 antibodies (middle panels), or an anti-HA antibody to detect SRSF3 (bottom panel). * indicates non-specific bands.