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. 2020 Jan 28;11(1):71. doi: 10.1038/s41419-020-2265-y

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a The FAM-labeled probes containing sequences of FoxO1-L-B (left) and FoxO1-R-B (right) and corresponding mutated probes (underlined; FoxO1-L-B-M and FoxO1-R-B-M), and the deleted sequences (line-through; FoxO1-L-B-D and FoxO1-R-B-D). b EMSA was carried out using normal or mutated FAM-labeled probes. The amount of DNA-protein complexes detected in FAM-labeled mutant probes was similar to that in FAM-labeled normal probes. c EMSA was carried out using FAM-labeled normal and deletion probes. Fewer DNA-protein complexes were detected with FAM-labeled deletion probes than the FAM-labeled normal probes followed by PARP1 incubation. d Schematic representation of the flanking restriction enhanced pulldown (FREP). A biotinylated DNA fragment is conjugated to streptavidin-coated magnetic Dynabeads (Invitrogen, Carlsbad, CA). This fragment is engineered to include the FoxO1-L-B or FoxO1-R-B specific (“bait”) sequence (black dashed box), flanked by restriction enzyme cleavage sites for BamH I proximally (gray dashed box) and EcoR I distally (gray box). DNA-beads are mixed with PARP1 protein. A free non-biotinylated FoxO1-L-B or FoxO1-R-B DNA fragment can be included in the control reaction at this stage as a specific competitor. Magnetic separation and wash remove non-DNA binding PARP1 protein. EcoR I digestion releases 3′ DNA end-binding PARP1, and BamH I digestion separates the sequence-specific FoxO1-L-B or FoxO1-R-B binding PARP1 from the 5′ DNA and Dynabeads. Western blotting identifies PARP1 binding to FoxO1-L-B or FoxO1-R-B. e The PARP1-DNA complexes cut with EcoR I and BamH I were detected by western blotting. The relative levels of FoxO1-bound PARP1 were presented as the ratio of FoxO1-bound PARP1 band intensity/PARP1 input band intensity when the value of PARP1 input band intensity was normalized as l. Lane 1: PARP1 inputs, lane 2: labeled FoxO1-L-B-beads, lane 3: PARP1 with labeled FoxO1-L-B-beads, lane 4: PARP1 with labeled FoxO1-L-B-beads and cold competitor (40-fold excess of free FoxO1-L-B-beads), lane 5: labeled FoxO1-R-B-beads, lane 6: PARP1 with labeled FoxO1-R-B-beads, lane 7: PARP1 with labeled FoxO1-R-B-beads and cold competitor (40-fold excess of free FoxO1-R-B-beads), lane 8: a labeled non-specific DNA sequence (NS-beads) and lane 9: PARP1 with labeled NS-beads.