Ab139470

To prepare the nuclear fractions, NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Fischer Scientific) were used according to the manufacturer’s instructions. The protein levels of the nuclear fractions were normalized to Lamin B1. The in vitro sumoylation assay was conducted using a commercial assay kit from Abcam (ab139470). The mixtures were set up in 20 μl of reaction buffer and incubated at 37 °C for 90 min. The reaction mixtures were subsequently separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. For in vitro translation, protein synthesis was achieved using the TNT T7/T3 Coupled Reticulocyte Lysate System (Promega, Fitchburg, WI, USA) according to the manufacturer’s instructions. In vitro translation was performed in a 25 μl reaction volume with 1 μg of construct. The reaction was incubated at 30 °C for 90 min.

Samples were extracted in RIPA buffer containing 1% SDS plus a protease inhibitor mix and 50 mM N‐ethylmaleimide (NEM) to block SUMO proteases. Lysates (500 μg of total protein) were subjected to IP using anti‐PSD95 antibody (3 μg/sample) crosslinked to paramagnetic beads (Bio‐Adembeads) overnight. The immune complexes were washed and eluted according to the manufacturer's instruction, and an equal amount of WT and AD immunoprecipitates were used in the in vitro SUMOylation assay (Abcam Ab139470). Where indicated, the GFP‐HDAC4^SD expressed in SH‐SY5Y or IgG‐control purified by IP with paramagnetic beads were added to the AD sample in the in vitro SUMOylation reaction. The products of the enzymatic reaction were processed by WB (8% SDS–PAGE under non‐reducing conditions) using anti‐SUMO2/3 (Cytoskeleton monoclonal ASM24), anti‐PSD95 (Cell Signaling polyclonal 3550) and anti‐HDAC4 (Abcam polyclonal Ab12172) antibodies to detect the immunoprecipitated proteins. SUMO‐RanGAP was used as a positive control of the enzymatic reaction.