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. 2021 Jul 29;20:98. doi: 10.1186/s12943-021-01394-8

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© The Author(s) 2021

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — Cytoplasmic circIKBKB facilitates IKKβ-mediated IκBα phosphorylation and nuclear circIKBKB inhibited IκBα feedback loop. a FISH (left) and qPCR from nuclear-cytoplasmic fractionation analysis of subcellular localization of circIKBKB. b circIKBKB pull-down following the mass spectrometry showed that circIKBKB interacted with IKKα/IKKβ/IKKγ and p65/p50/IκBα complex. c RNA pull-down assay analysis of cytoplasmic and nuclear circIKBKB-interacting proteins (“probe” stands for circIKBKB probe, “Ctrl” stands for control probe). d RNA pull-down assay showing that circIKBKB only bound to active IKKβ. e RIP assay analysis of the binding region of circIKBKB with IKKβ or p65. f Schematic illustration of Flag-tagged full-length p65 and three truncated p65 fragments (left) and RNA pull-down assay analysis(right) of interaction between circIKBKB with p65 fragments. g IP (upper) and WB (lower) analysis showing that cytoplasm circIKBKB facilitated IKKβ/p65 interaction and IKKβ-mediated IκBα phosphorylation in a dose dependent manner. GAPDH served as loading control. h IP (upper) and WB (lower) analysis showing that nuclear circIKBKB inhibited IκBα feedback loop. P84 served as loading control. i Chromatin fraction and WB analysis of DNA-bound NF-κB in the indicated cells. H3 served as a loading control. j EMSA analysis of NF-κB activity in the indicated MDA-MB-231 cells treated with TNF-α (10 ng/ml) for the indicated times. Each error bar represents the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001