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. 2016 Feb 29;44(6):2475–2490. doi: 10.1093/nar/gkw118

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© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 5. — QKI-7 specifically interacts with PAPD4 through its unique C-terminal region to induce mRNA polyadenylation. (A) HEK293T cells were co-transfected with pCMV-5×Myc-PAPD4 and either pCMV-5×Flag-GST (lanes 1 and 4), pCMV-5×Flag-CPEB (lanes 2 and 5) or pCMV-5×Flag-QKI-7 (lanes 3 and 6). Cell extracts were subjected to immunoprecipitation (IP) using the anti-Flag antibody. The immunoprecipitates (lanes 4–6) and inputs (lanes 1–3) were analyzed by Western blotting with the indicated antibodies. (B) HEK293T cells were co-transfected with pCMV-5×Myc-PAPD4 and either pCMV-5×Flag-GST (lanes 1 and 7), pCMV-5×Flag-QKI-7 (lanes 2 and 8), pCMV-5×Flag-QKI-7(1–205) (lanes 3 and 9), pCMV-5×Flag-QKI-7(1–235) (lanes 4 and 10), pCMV-5×Flag-QKI-7(1–265) (lanes 5 and 11) or pCMV-5×Flag-QKI-7(1–295) (lanes 6 and 12). Cell extracts were subjected to IP using the anti-Flag antibody. The immunoprecipitates (lanes 7–12) and inputs (lanes 1–6) were analyzed by Western blotting with the indicated antibodies. (C) HEK293T cells were co-transfected with pCMV-5×Myc-PAPD4 and either pCMV-5×Flag-GST (lanes 1 and 5), pCMV-5×Flag-QKI-5 (lanes 2 and 6), pCMV-5×Flag-QKI-6 (lanes 3 and 7) or pCMV-5×Flag-QKI-7 (lanes 4 and 8). Cell extracts were subjected to IP using the anti-Flag antibody. Immunoprecipitates (lanes 5–8) and inputs (lanes 1–4) were analyzed by Western blotting with the indicated antibodies. (D) HEK293T cells were co-transfected with pCMV-5×Myc-QKI-7 and either pCMV-5×Flag-GST (lanes 1 and 5), pCMV-5×Flag-PAPD4 (lanes 2 and 6), pCMV-5×Flag-PAPD5 (lanes 3 and 7) or pCMV-5×Flag-PAPD7 (lanes 4 and 8). Cell extracts were subjected to IP using anti-Flag antibody. Immunoprecipitates (lanes 5–8) and inputs (lanes 1–4) were analyzed by Western blotting with the indicated antibodies. (E) HEK293T cells were co-transfected with pCMV-5×Myc-QKI-7 and either pCMV-5×Flag-GST (lanes 1 and 6), pCMV-5×Flag-PAPD4 (lanes 2 and 7), pCMV-5×Flag-PAPD4(1–141) (lanes 3 and 8), pCMV-5×Flag-PAPD4(142–484) (lanes 4 and 9) or pCMV-5×Flag-PAPD4(1–207) (lanes 5 and 10). Cell extracts were subjected to IP using anti-Flag antibody. Immunoprecipitates (lanes 6–10) and inputs (lanes 1–5) were analyzed by western blotting with the indicated antibodies. (F) HEK293T cell extracts were subjected to IP using anti-PAPD4 or normal goat IgG as a non-specific control. The immunoprecipitaes (lanes 2 and 3) and input (lane 1) were analyzed by Western blotting with the indicated antibodies. (G) HEK293T cells were co-transfected with the pFlag-CMV5/TO-BGG (1–39)-MS2bs reporter plasmid, the pCMV-5×Flag-EGFP reference plasmid, either pMS2–5×Myc-GST (lanes 1 and 3) or pMS2–5×Myc-QKI7 (lanes 2 and 4), and either Luciferase siRNA (lanes 1 and 2) or PAPD4 siRNA (lane 3 and 4). BGG(1–39)-MS2bs mRNA was detected by Northern blot analysis. (H) BGG(1–39)-MS2bs mRNA length-distribution was visualized as in Figure 1D. (I) Protein expression was analyzed by Western blotting with the indicated antibodies.