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. 2016 Feb 9;44(6):2846–2858. doi: 10.1093/nar/gkw027

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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. — Silencing circ-Foxo3 enhanced cell cycle entry and decreased the interaction between p21 and CDK2. (A) Lysates prepared from subconfluence NIH3T3 cells transfected with circ-Foxo3 siRNA or a control oligo were subject to western blotting. Silencing circ-Foxo3 did not change expression of p21 and CDK2. (B) The lysates were subject to IP with anti-p21 antibody followed by western blotting. p21 precipitation pulled-down less CDK2 in the circ-Foxo3 siRNA-transfected cells relative to the control. (C) The lysates were subject to IP with anti-CDK2 antibody, followed by western blotting. CDK2 precipitation pulled-down less p21, but more cyclin A and cyclin E in the siRNA-transfected cells compared to the control. (D) Silencing circ-Foxo3 did not change expression of cyclin A and cyclin E. (E) Cyclin A and cyclin E precipitations pulled down more CDK2 in the circ-Foxo3 siRNA-transfected cells. (F) The lysates were subject to native gradient gel electrophoresis followed by western blotting probed with antibodies against p21 or CDK2. (G) Cell lysates prepared from p21 siRNA- and a control oligo-transfected NIH3T3 cells were subject to immunoprecipitation with anti-rabbit IgG, mouse IgG, p21 and Cdk2 antibodies, followed by real-time PCR. Anti-p21 antibody pulled-down less circ-Foxo3 in the p21 siRNA-transfected cells. **P < 0.01. Error bars, SD (n = 4). (H) The lysates were hybridized with circ-Foxo3 probe or a control oligo for RNA pull-down assays. Real-time PCR showed that the circ-Foxo3 probe pulled down same levels of circ-Foxo3 in the cells transfected with p21 siRNA. Error bars, SD (n = 4). (I) Silencing p21 did not affect CDK2 expression. However, anti-p21 antibody pulled-down less Cdk2 and p21 in the p21 siRNA-transfected cells. Anti-CDK2 antibody pulled-down less p21, but the same amount of Cdk2 in the p21 siRNA-transfected cells. (J) Silencing CDK2 did not affected circ-Foxo3 levels. (K) Anti-CDK2 antibody pulled-down less circ-Foxo3 in the Cdk2 siRNA-transfected cells. **P < 0.01. Error bars, SD (n = 4). (L) Silencing CDK2 did not affect p21 expression. However, anti-CDK2 antibody pulled-down less Cdk2 and p21 in the CDk2 siRNA-transfected cells. Anti-p21 antibody pulled-down less CDK2, but the same amount of p21 in the CDK2 silencing cells.