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. 2015 Jul 27;4(7):e161. doi: 10.1038/oncsis.2015.20

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Copyright © 2015 Macmillan Publishers Limited

Oncogenesis is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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AFF3 overexpression alters nuclear speckles and pTEFb. (a) Lines 1 and 2: H295R cells were transfected with AFF3-Flag vector. The AFF3 protein was revealed with an anti-Flag antibody and nuclear speckles were detected using an anti-SC35 antibody. After 8 h of expression of the vector, AFF3 colocalized with SC35 in nuclear speckles; after 24 h, the distribution of SC35 was altered. Lines 3 and 4: CDK9 and cyclin T1 protein were detected using anti-CDK9 and anti-cyclin T1 antibodies. In H295R cells, CDK9 and cyclin T1 colocalized with SC35 in nuclear speckles. Lines 5 and 6: H295R cells were transfected with AFF3-Flag vector. AFF3 overproduction resulted in CDK9 and cyclin T1 being found colocalized with AFF3. The white dashed rectangle in the merge column represents the enlarged area used for colocalization analysis using ImageJ software. Pearson's colocalization coefficients (PCC) were determined with JACoP plugin⁴⁹ in cells from different experiments (n⩾10). t-test was used to evaluate the difference between PCC for AFF3/SC35 colocalization analysis after 8 and 24 h of transfection (lines 1 and 2). (b) Protein lysates from H295R cells overexpressing AFF3-Flag or Ctr vectors were immunoprecipitated with Anti-Flag M2 Affinity Gel and analyzed by western blot using the indicated antibodies. Overproduced AFF3 associated with CDK9 and Cyclin T1. Protein lysates from H295R cells overexpressing AFF3-Flag or silenced for AFF3 by siAFF3 were immunoprecipitated with anti-CDK9 antibodies and analyzed by western blot using the indicated antibodies. CDK9 associated with overproduced and endogenous AFF3. AFF3 silencing in H295R cells confirmed the specificity of the protein band of endogenous AFF3.