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. 2022 Dec 9;12:21343. doi: 10.1038/s41598-022-25665-6

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© The Author(s) 2022

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 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/.

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Specificity of SUMO MAbs by immunoblot. (a) U2OS cells with the ability to express siRNA-resistant FLAG-SUMO1 treated with non-targeting control siRNA (siNTC), or siRNA to SUMO1 (siSUMO1) or treated with siSUMO1 and with doxycycline for 48 h to induce FLAG-SUMO1. Lysates were separated by 4–20% SDS-PAGE and immunoblotted with indicated MAbs. All blots are shown at 1 min exposure. (b) Heat map of total SUMO1 signal (10 kDa to well front) expressed as a % relative to siNTC. Deep red indicates high rescue of the siRNA-resistant FLAG-SUMO1. N = 3. (c) U2OS cells able to express siRNA-resistant forms of FLAG-SUMO2 and FLAG-SUMO3 were treated with siRNA to both SUMO2 and SUMO3 and probed with the SUMO2/3 antibodies shown. (d) Heat map relative quantification of SUMO2/3 signal (10 kDa to well front) detected by antibodies raised to SUMO2/3. N = 3. (e) Lysates from 2c probed with SUMO4 MAbs. (f) Heat map relative quantification of the SUMO2/3 signal (10 kDa to well front) detected with SUMO4 Mabs. N = 3.