Figure 4. GluK2 is not SUMO1-conjugated in vivo and in vitro.
(A) SDS-PAGE (4–12%) followed by Anti-GluK2 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 brain. The presence of GluK2 in both WT and KI eluates indicates non-specific binding of GluK2 to the affinity matrix. (B) Representative SDS-PAGE (10%) followed by anti-HA and anti-GluK2 Western blot analysis of input and eluate fractions from anti-GluK2 and anti-IgG immunoprecipitations in the presence of 20 mM NEM from WT and His6-HA-SUMO1 KI brains. GluK2 is specifically enriched in both WT and KI samples after affinity purification using anti-GluK2 but not in control experiment conducted with rabbit IgG (upper panel). However, anti-HA Western blot does not reveal any band that could have corresponded to SUMO1-GluK2 (lower panel). (C) Representative SDS-PAGE (10%) followed by anti-HA and anti-GluK2 Western blot of input and eluate fractions from anti-HA immunopreciptation in the presence of 20 mM NEM from HEK cells overexpressing HA-SUMO1 and GluK2, alone or in combination. Anti-HA Western blot analysis confirms the enrichment of HA-SUMO1 conjugates (lower panel) but no SUMO1-GluK2 band is observed in the eluate fractions (upper panel). Images are representatives of at least three independent experiments.