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. 2021 Nov 25;12:6896. doi: 10.1038/s41467-021-27210-x

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© The Author(s) 2021

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. 5 — a 753b degrades WT BCL-xL and K20-only and K87-only BCL-xL mutants, but not K16-only BCL-xL mutant, in 293T cells. K16-only, K20-only, and K87-only represent that all the surface lysines on BCL-xL are mutated to arginines except K16, K20, and K87, respectively. Representative immunoblots of Flag-BCL-xL WT and mutants are shown in the top panel. Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing DMSO- to 753b-treated samples. *P < 0.05; **P < 0.01; P values were 0.0030, 0.0070, and 0.035 for WT, K20-only, and K87-only BCL-xL, respectively. b 753b degrades WT BCL-2 and K22R BCL-2 mutant, but not K17R and K17/22R BCL-2 mutants, in 293T cells. K17R and K22R represents that K17 and K22 on BCL-2 are respectively mutated to arginine; K17/22R means both K17 and K22 on BCL-2 are mutated to arginine. Representative immunoblots of Flag-BCL-2 WT and mutants are shown in the top panel. The quantification of the relative Flag-BCL-xL or Flag-BCL-2 protein content in the immunoblots is presented as mean values ± s.e.m. (n = 3 biologically independent experiments) in the bottom panel. Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing DMSO- to 753b-treated samples. **P < 0.01; P values were 0.0043 and 0.0020 for WT and K22R BCL-2 mutant, respectively. c 753b induces polyubiquitination of WT, K20-only, and K87-only BCL-xL, but not K16-only BCL-xL in 293 T cells. d 753b induces polyubiquitination of WT and K22R BCL-2, but not K17R and K17/22R BCL-2 in 293T cells. For c, d 293T cells were co-transfected as indicated with Flag-BCL-xL WT or mutants and HA-tagged ubiquitin (HA-Ub) plasmids (c) or Flag-BCL-2 WT or mutants and HA-Ub plasmids (d). After 36 h, cells were pre-treated with MG132 (10 µM) for 2 h and then treated with or without 753b (0.1 µM for BCL-xL and 1 uM for BCL-2) for 5 h. The Flag-tagged proteins were immunoprecipitated (IP) and immunoblotted with HA or Flag antibody. The input results are shown in Supplementary Fig. 9. Data are a representative of two independent experiments. Source data are provided as a Source data file.