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. 2020 Aug 12;11(8):683. doi: 10.1038/s41419-020-02848-5

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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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Fig. 5 — a Pattern diagram indicating the distribution of all lysine residues of 48-kD PAX6. b 293T cells were co-transfected with HA-tagged SUMO1 with the indicated wild-type PAX6 and PAX6 variants, containing one or two adjacent lysine (K) residues mutated to arginine (R) residues (K28R, K53R, K69R, K89R, K100/K105R, K148R, K159R, K221/223R, K241R, K260R, K278R, or K284R). Cell lysates were prepared under denaturing conditions and subjected to immunoprecipitation with anti-Flag antibody, followed by western blot using anti-HA, anti-Flag, or anti-GAPDH antibodies. c 293T cells were co-transfected with Flag-tagged SUMO1 with the indicated wild-type PAX6 and PAX6 variants, containing all lysine-to-arginine mutations, but one or two adjacent lysine (K) sites remained (K28(1), K53(1), K69(1), K89(1), K100(1), K105(1), K148(1), K159(1), K221/223(2), K241(1), K260(1), K278(1), or K284(1)). Cell lysates were prepared under denaturing conditions and subjected to immunoprecipitation with anti-Flag antibody, followed by western blot using anti-HA, anti-Flag, or anti-GAPDH antibodies. Showing K53(1) and K89(1) restored the most apparent expression of SUMO1-modified PAX6. d Flag-tagged plasmids encoding wild-type PAX6 (WT) and SUMO1-modified sites that mutated PAX6 (K53R, K89R, and K53/89R) were constructed. 293T cells were co-transfected with Flag-tagged plasmids encoding PAX6 WT, K53R, K89R, and K53/89R, HA-tagged SUMO1 as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody and analyzed by western blot with anti-Flag, anti-HA, or anti-GAPDH antibodies.