Figure 6. SUMO1 conjugates are mainly in nuclear fractions.
SDS-PAGE (4–12%) followed by Western blot analysis of subcellular fractions performed in the presence of 20 mM NEM of His6-HA-SUMO1 KI and WT mouse brain using anti-SUMO1 (upper two panels) and anti-HA antibodies (below), and antibodies to GluN1 and synaptophysin to validate the fractionation procedure (lower two panels). Bracket indicates specific SUMO1 substrates while stars indicate non-specific SUMO1 bands present in both His6-HA-SUMO1 KI and SUMO1 KO. H, homogenate; P1, nuclear pellet; S1, supernatant after P1 sedimentation; P2, crude synaptosomal pellet; S2, supernatant after P2 sedimentation; LP1, lysed synaptosomal membranes; LS1, supernatant after LP1 sedimentation; SPM, synaptic plasma membranes. Images are representatives of at least three independent experiments.