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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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SUMO MAbs as SUMOylation enrichment tools. (a) U2OS cells with the ability to express siRNA-resistant FLAG-SUMO1 treated with siSUMO1 and doxycycline for 48 h before denaturing lysis and immunoprecipitation with the indicated Mabs. Immunoprecipitated material was divided in two and probed with anti-FLAG or anti-RANGAP1 antibodies. The anti-FLAG panel is from a 30-s exposure, while the anti-RANGAP1 blot was exposed for 1 min. The input for RANGAP1 detects both the unmodified and SUMO1 conjugated species (~ 70 and 100 kDa, respectively). (b) Quantitation of FLAG-SUMO1 IP signal (from 70 kDa to the well front) from 6 experiments. The enrichment is shown as a mean % relative to the M2 anti-FLAG IP from the same cell lysate as an internal control for each experiment. Error bars show S.E.M. (c) Quantitation of the mean RANGAP1 IP signal (100 kDa species) by antibodies raised to SUMO1 relative to RANGAP1 precipitation by anti-FLAG, M2. Error bars show S.E.M. N = 6. (d) Immunoprecipitation of FLAG-SUMO2 by anti-FLAG and MAbs raised to SUMO2/3 from U2OS cells with the ability to express FLAG-SUMO2 (treated as for a). (e) Quantification of the mean FLAG-SUMO2 IP signal by antibodies raised to SUMO2/3 as a % of those precipitated by anti-FLAG M2, from 5 experiments. Error bars show S.E.M. (f) Immunoprecipitation of FLAG-SUMO2 by M2 and SUMO4 MAbs (treated as for a but using SUMO4 MAbs). (g) Immunoprecipitates by anti-FLAG and anti-SUMO2/3 MAbs, from (d), probed for KAP1. The approximate location of mono, di and tri-SUMO modified KAP1 is shown. (h) Quantification of the mean relative KAP1 enrichment by anti-SUMO2/3 MAbs relative to anti-FLAG M2, from at least 3 experiments. Error bars show S.E.M. (i) Relative IP enrichment for FLAG-SUMO1 (total SUMOylation) versus RANGAP1-SUMO1 enrichment (from data in b,c). The IP efficiency of FLAG M2 is set at 100% for both conditions. (j) Relative IP enrichment of FLAG-SUMO2 and KAP1 by SUMO2/3 antibodies (from data in e,f) relative to anti-FLAG M2.