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. 2015 May 24;43(12):5868–5879. doi: 10.1093/nar/gkv541

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© The Author(s) 2015. 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/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — AFF1 and AFF4 reside in different SECs. (A,B) Nuclear extracts (NEs) derived from HeLa cells co-transfected with the indicated cDNA constructs were subject to anti-Flag immunoprecipitation (IP). The precipitates and NEs were examined by western blotting (WB) for the indicated proteins. (C,D) HeLa cells were transfected with plasmid vectors expressing the indicated Flag-tagged WT AFF1 (C), AFF4 (D) or their mutant derivatives. Anti-Flag immunoprecipitates from NE were analyzed by WB for the various proteins as marked on the left, with the star (*) in C denotes the position of two minor truncated AFF1 bands. (E) Whole cell extracts of the indicated HEK293-based cell lines were analyzed by WB. Both 293-F9 and 293-F9H4 stably expressed CDK9-F, while the latter also expressed AFF4-HA upon induction by doxycycline (Dox). (F,G) CDK9-F, AFF4-HA and their associated factors were isolated through sequential immunoprecipitations (anti-HA and then anti-Flag in F; anti-Flag and then anti-HA in G) from NEs of 293-F9H4 cells upon induction of AFF4-HA expression. The immunoprecipitates were analyzed by WB for the proteins indicated. NEs of 293-F9 cells were used in parallel procedures as controls.