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. 2020 Jan 21;10:766. doi: 10.1038/s41598-020-57698-0

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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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SENP2 negatively regulates NF-κB activation through the modulation of IκBα sumoylation. (A) Either Scramble shRNA or SENP2-shRNA were transiently transfected in RPMI8226 cells. After 48 hours, western blot analysis was performed to detect SENP2 protein levels. Here, GAPDH serves as a loading control. (B) MM cell lines, RPMI8226 and MM.1S were transfected with either scramble shRNA or SENP2-shRNA for 48 hours, then western blot analysis was performed to detect SUMO2 protein levels. Here, GAPDH serves as a loading control. (C) EMSA assay was performed to detect NF-κB binding to its consensus binding sites on biotin labelled DNA probe using cell lysates of RPMI8226 cells transfected with either Scramble shRNA or SENP2-shRNA for 48 hours and either untreated or treated with bortezomib 25 nM. Here, OCT1 serves as an internal control for EMSA assay. (D) NF-κB activation was analysed by western blotting analysis using cell lysates of RPMI8226 cells transfected with either scramble shRNA or SENP2-shRNA for 48 hours and followed by probing with antibodies against p-p65, p65, p-IκBα and IκBα. Here, GAPDH serves as a loading control. (E) Either scramble shRNA or SUMO2-shRNA were transiently transfected in RPMI8226 cells. After 48 hours, western blot analysis was performed to detect SUMO2 protein levels. Here, GAPDH serves as a loading control. (F) EMSA assay was performed to detect NF-κB binding to its consensus binding sites on biotin labelled DNA probe using cell lysate of RPMI8226 cell lysates transfected with either Scramble shRNA or SUMO2-shRNA for 48 hours. Here, OCT1 serves as an internal control for EMSA assay. (G) Reciprocal immunoprecipitation analysis using anti-IκBα and anti-SUMO2 antibodies from cell extracts of RPMI8226 cells transfected with Scramble shRNA or SENP2-shRNA for 48 hours.