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. 2013 Feb 1;10(2):228–244. doi: 10.4161/rna.23018

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Copyright © 2013 Landes Bioscience

This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.

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graphic file with name rna-10-228-g8.jpg — Figure 8. The NOT10-NOT11 complex is conserved in human cells. (A) Human CNOT1 consists of an N-terminal (NOT1-N), middle (NOT1-M), and C-terminal (NOT1-C) region. The POP2-binding domain adopts an MIF4G fold and is termed the CNOT1 MIF4G domain.¹⁵^,¹⁶ The CNOT1-C region harbors a conserved NOT1 homology domain. (B) GFP-tagged CNOT1 (full-length or fragments) or GFP-CNOT11 were coexpressed with HA-tagged CNOT10 in HEK293 cells. The GFP-tagged proteins were immunoprecipitated from RNase A-treated cell lysates using anti-GFP antibodies. GFP-tagged MBP served as a negative control. Inputs (1.25% for the GFP-tagged proteins or 0.5% for HA-tagged proteins) and immunoprecipitates (10% for the GFP-tagged proteins or 25% for HA-tagged proteins) were analyzed by western blotting. (C) HEK293 cells were cotransfected with a mixture of two plasmids: one expressing GFP-CNOT1 and one expressing HA-CNOT11. In addition, where indicated, the transfection mixtures contained a third plasmid expressing HA-CNOT10 (lanes 3, 4, 7 and 8). Cell lysates were immunoprecipitated using polyclonal anti-GFP antibodies. The inputs and immunoprecipitates were analyzed by western blotting as described in panel B. (D) Interaction of GFP-tagged CNOT11 (full-length or DUF2363 domain) with HA-tagged CNOT10 in HEK293 cells.