Fig 1: N-ethylmaleimide (NEM) dependent retention of SUMO-modified Arc. Coimmunoprecipitation was carried with and without the addition of the cysteine (SUMO) protease inhibitor NEM to the lysis and immunoprecipitation buffer. (A) Detection of enhanced Arc SUMOylation during LTP is NEM-dependent. The ratio of upper to lower Arc immunoreacitive bands was determined in Arc immunoprecipitates. Bar graphs show fold change in HFS-treated dentate gyrus compared to control. n = 5; Student’s t-test, *P < 0.05. (B) Immunoprecipitation of Arc, SUMO1 and SUMO2/3 was performed in the presence or absence of NEM and precipitates were immunoblotted for Arc. Representative blot from a single gel. A 65 kDa immunoreactive Arc band (arrow) was reliably detected only in NEM-processed samples. The non-covalent interaction of Arc with SUMO1 and SUMO2/3 was also NEM-dependent. (C) Arc immunoblots of dentate gyrus lysate (input) samples. NEM treatment tended to increase Arc immunoreactivity but there was no difference in fold increases in Arc with and without NEM (n = 5, P > 0.05). (D) SENP1 enzymatic treatment of Arc immunoprecipitate abolished 65 kDa SUMO-Arc. In this immuno-precipitation performed with a polyclonal antibody from Synaptic Systems, an additional 75 kDa Arc immunoreactive band was detected by the monoclonal mouse Arc C7 antibody. However, the 75 kDa band could not be validated in the reverse immunoprecipitation or by SUMO1 immunoprecipitation.
Fig 2: Enhanced Arc SUMOylation in the synaptoneurosome compartment during LTP in vivo. (A) Representative Arc immunoblot performed in dentate gyrus whole lysate and fractionated synaptoneurosome samples. (B) Fold change in SUMOylated Arc based on the ratio of the upper and lower Arc immunoreactive bands. Two dentate gyri were pooled each experiment. n = 4; Student’s t-test, *P < 0.05. (C) SUMO1immuno-precipitation (mouse monoclonal) followed by Arc immunoblotting confirmed Arc SUMOylation in synaptoneurosomes. Unmodified Arc is also detected in the SUMO1 pellet. (D) SENP1 digestion removed SUMOylated Arc. The heavy band detected in the Arc and SUMO1 immunoprecipitate is absent in SENP1 treated samples. Arc was immunoprecipitated with mouse monoclonal Arc C7 antibody. #The polyclonal rabbit Arc antibody (Synaptic Systems) used for immunoblotting detects a spurious band in the lysate that is absent in the Arc pellet. *Bands due to SENP1 enzyme in the blot; right lane shows SENP1 enzyme loaded alone. 6% polyacrylamide gel.
Supplier Page from R&D Systems, a Bio-Techne Brand for His6 SENP1 Catalytic Domain Protein
Available conjugates: Sizes Available: 50 ug