Figure 1. Zbtb20 is a SUMO1 substrate in vivo and in vitro.

(A) SDS-PAGE (4–12%) followed by anti-Zbtb20 Western blot analysis of input and HA peptide eluate fractions from anti-HA immunoprecipitation in the presence of 20 mM NEM from WT and His₆-HA-SUMO1 KI brains. Zbtb20 is specifically enriched in KI eluate (bracket), indicating that Zbtb20 is a SUMO1 target in vivo. (B) Representative SDS-PAGE (8%) followed by anti-Zbtb20, SUMO1, and HA Western blot analysis of input and eluate fractions of anti-Zbtb20 and anti-IgG immunopurifications from WT and His₆-HA-SUMO1 KI brains in the presence of 20 mM NEM. Anti-Zbtb20 Western blot confirm the enrichment of Zbtb20 in both WT and KI brain samples solely when anti-Zbtb20 antibody is used (upper panel, brackets). Anti-SUMO1 Western blot analysis revealed a shifted band corresponding to SUMO1-Zbtb20 in both WT and KI (middle panel, black arrow). Anti-HA Western blot revealed a shifted band corresponding to His₆-HA-SUMO1-Zbtb20 solely in KI eluate (lower panel, black arrow). (C) SDS-PAGE (10%) followed by Western blot analysis of input and eluate fractions of anti-HA immunoprecipitation in the presence or absence of 20 mM NEM from HEK cells overexpressing HA-SUMO1 and Myc-Zbtb20-WT, alone or in combination. Anti-HA Western blot confirms enrichment of HA-SUMO1 conjugates in the presence of NEM while all SUMO1 conjugates (with the exception of RanGAP1) are lost when NEM is omitted (lower panel). Anti-Myc Western blot analysis confirms the co-enrichment of Myc-Zbtb20-WT in the presence of NEM and when co-expressed with HA-SUMO1. (D) SDS-PAGE (10%) followed by Western blot analysis of input and eluate fractions of anti-HA immunoprecipitation in the presence or absence of 20 mM NEM from HEK cells overexpressing HA-SUMO1 and Myc-Zbtb20-2KR, alone or in combination. Anti-HA Western blot confirms enrichment of HA-SUMO1 conjugates in the presence of NEM while all SUMO1 conjugates (with the exception of RanGAP1) are lost when NEM is omitted (lower panel). Anti-Myc Western blot analysis shows that Myc-Zbtb20-2KR is not co-enriched in the presence of NEM and when co-expressed with HA-SUMO1 (upper panel). Images are representatives of at least three independent experiments.

DOI: http://dx.doi.org/10.7554/eLife.26338.002

Figure 1—figure supplement 1. SUMO1 levels in the nuclei of His₆-HA-SUMO1 KI neurons (also relates to Figures 2–8, which include results obtained with the His₆-HA-SUMO1 KI).

Primary hippocampal neurons from WT (12 days in vitro) and His₆-HA-SUMO1 KI mice (11 days in vitro) were fixed, permeabilised with Triton X-100, and immunolabelled with antibodies against SUMO1 and Map2, as well as DAPI to label nuclei. Representative images are shown of a WT neuron (A) and a His₆-HA-SUMO1 KI neuron (B). (C) Graph showing the nuclear anti-SUMO1 labelling intensity of neuronal nuclei from WT and His₆-HA-SUMO1 KI mice (WT, N = 2 mice, 28 neurons; His₆-HA-SUMO1 KI, N = 4 mice, 56 neurons). The scatter plots show the data from individual cells while the horizontal line shows the mean nuclear anti-SUMO1 intensity from each genotype (WT, 54.8 AU, His₆-HA-SUMO1 KI, 35 AU). Scale bar = 10 µm.

DOI: http://dx.doi.org/10.7554/eLife.26338.003

Figure 1—figure supplement 2. Characterisation of the anti-HA immunoprecipitation and of the anti-Zbtb20 antibody.

(A) Characterisation of the anti-HA immunoprecipitation in the His₆-HA-SUMO1 KI line. SDS-PAGE (4–12%) followed by Western blot analysis of input and HA peptide eluate fraction using anti-HA antibody from anti-HA IP from WT and His₆-HA-SUMO1 KI brains in the presence of 20 mM NEM. Anti-HA signal is observed solely in the KI eluate, indicating the specific and strong enrichment of His₆-HA-SUMO1 species. (B) Characterization of the specificity of anti-Zbtb20 antibody. Total brain homogenate from embryonic day E18 mice being (WT, +/+; heterozygous Zbtb20 KO, +/−, and homozygous Zbtb20 KO) analysed by SDS-PAGE (10%) and Western blotting with anti-Zbtb20 antibodies. Western blot analysis using anti-Actin antibody ensured equal loading of lanes.

DOI: http://dx.doi.org/10.7554/eLife.26338.004