Figure 2. Synapsin1a is not SUMO1-conjugated in vivo and in vitro.
(A) SDS-PAGE (4–12%) followed by Western blot analysis using anti-synapsin1 antibody 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. The presence of synapsin1 in both WT and KI eluates indicates non-specific binding of synapsin1a to the affinity matrix. (B) Representative SDS-PAGE (10%) followed by Western blot analysis of input and eluate fractions of anti-synapsin1 and anti-IgG immunopurifications in the presence of 20 mM NEM from WT and His6-HA-SUMO1 KI brains. Anti-synapsin1 Western blot confirms enrichment of synapsin1 after affinity purification using synapsin1 antibody and not using nonrelated IgG (black arrow). However, anti-HA Western blot does not detect any SUMO1-synapsin1 shifted band. (C) SDS-PAGE (10%) followed by Western blot analysis of input and eluate fractions of anti-HA immunoprecipitation in the presence of 20 mM NEM from HEK cells overexpressing HA-SUMO1 and GFP-synapsin1a, alone or in combination. While anti-HA Western blot confirms enrichment of HA-SUMO1 species (lower panel), synapsin1a is not detected in the eluate samples (upper panel). Images are representatives of at least three independent experiments.