sc 9060  (Santa Cruz Biotechnology)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Promyelocytic Leukemia Protein (PML) Requirement for Interferon-induced Global Cellular SUMOylation *

doi: 10.1074/mcp.RA117.000447

Figure Lengend Snippet: PML is required for IFN-enhanced SUMOylation. A, B, wt MEFs and PML−/− MEFs were untreated or treated with murine IFNα for short (0.75 and 1 h) (A) or long (16 h) (B) periods and their extracts were analyzed by Western blotting for SUMO2/3, SUMO1, PML or Actin. C, D, PML−/− MEFs were transduced with HIV-1 derived lentiviral vectors expressing each human PML isoform (C) (PMLI, PMLII, PMLV, PMLVI or PMLVII), (D) (PMLIII or PMLIV). Two days post-transduction, PML expressing cells were untreated or treated with murine IFNα for 16 h and their extracts were analyzed by Western blotting for SUMO2/3, PML and Actin.

Article Snippet: Mouse monoclonal anti-PML (sc-966) antibody and rabbit polyclonal antibodies raised against PML (Sc-5621), STAT1 (sc-345), STAT1 phosphotyrosine 701 (sc-7988), PKR (sc-707), p53 (sc-126), and SUMO1 (sc-9060) as well as peroxidase-coupled secondary antibodies were from Santa-Cruz (Dallas, TX).

Techniques: Western Blot, Transduction, Derivative Assay, Expressing

Journal: Frontiers in Synaptic Neuroscience

Article Title: Dynamic Arc SUMOylation and Selective Interaction with F-Actin-Binding Protein Drebrin A in LTP Consolidation In Vivo

doi: 10.3389/fnsyn.2017.00008

Figure Lengend Snippet: SUMOylation of ectopically expressed activity-regulatedcytoskeleton-associated protein (Arc) and in vitro SUMOylation assay. (A) Ectopically expressed Arc is SUMOylated and forms non-covalent complexes with small ubiquitin-like modifier 1 (SUMO1) and SUMO2/3 moieties in HT-1080 fibrosarcoma cell lines. Extract of cells coexpressing Arc with His6-tagged SUMO 1, 2 or 3 were subjected to nickel affinity chromatography and immunoblotted for Arc. A high molecular mass band at ~65 kDa (arrow) corresponding to the covalent addition of single His6-tagged SUMO1, SUMO2, or SUMO3 to Arc was detected. The molecular mass of His6-SUMOylated Arc is higher than physiologically SUMOylated Arc, due to histidine tag residues. The presence of non-modified Arc protein indicates non-covalent complex formation of Arc with His-SUMO modified proteins or free SUMO. (B) Arc was immunoprecipitated from HT-1080 cells expressing non-tagged Arc protein and in vitro SUMOylation was performed with SUMO activating (E1) and conjugating (E2) enzymes in the presence or absence of SUMO1. Upon addition of SUMO1, an Arc immunoreactive band at 65 kDa was detected, corresponding to single SUMO1ylation of Arc. Equal amounts of protein were loaded. No bands were observed in the gel above the 100 kDa marker.

Article Snippet: Antibodies: Arc C7 mouse monoclonal (1:200, sc-17839), Arc H300 rabbit polyclonal (1:200, sc-15325), Cofilin (1:500, sc-32158), Drebrin A (1:200, #sc-374269), Dynamin 2 (1:1000, sc-6400), GAPDH (1:5000, sc-32233), Histone 1 (1:500, sc-10806), rabbit polyclonal SUMO1 (1:1000, sc-9060), mouse monoclonal SUMO1 (1:1000, sc-5308), SUMO2/3 (1:1000, sc-32873), normal mouse IgG and rabbit IgG were from Santa Cruz Biotechnology.

Techniques: Activity Assay, In Vitro, Affinity Chromatography, Modification, Immunoprecipitation, Expressing, Marker

Journal: Frontiers in Synaptic Neuroscience

Article Title: Dynamic Arc SUMOylation and Selective Interaction with F-Actin-Binding Protein Drebrin A in LTP Consolidation In Vivo

doi: 10.3389/fnsyn.2017.00008

Figure Lengend Snippet: SUMO1ylation of endogenous Arc following in vivo long-term potentiation (LTP) induction. (A) Time course plots of medial perforant path-dentate gyrus evoked field excitatory postsynaptic potential (fEPSP) recorded before and after high-frequency stimulation (HFS; indicated by arrows). Values are mean ± SEM of the maximum fEPSP slope expressed in percent of baseline. Test pulses were applied at a 0.033 Hz. HFS (3 × 400 Hz bursts) as indicated by the arrows. Rats were killed at 1 h and 3 h post-HFS (stippled line) and dentate gyrus was microdissected for biochemical analysis. (B–F) Coimmunoprecipitation analysis of dentate gyrus extracts. Bar graphs (left panels) based on densitometric analysis of indicated bands expressed as fold change in the ipsilateral HFS-treated dentate gyrus relative to the contralateral, non-stimulated side. Right panels show representative immunoblots from LTP experiments and naïve dentate gyrus. IgG lane is IgG-coupled beads plus lysate from HFS-treated dentate gyrus. (B) SUMO1 immunoprecipitation (rabbit polyclonal) followed by immunoblotting with Arc C7 antibody. Quantification of upper band (SUMOylated Arc). (C) Arc immunoprecipitation followed by SUMO1 immunoblot. Quantification of upper band (SUMO-Arc). The band at 50 kDa is non-specific (IgG). (D) SUMO2/3 immunoprecipitation followed by Arc immunoblot. (E) Arc immunoprecipitation (H300) followed by Arc (C7) immunoblot. Fold change in Arc SUMOylated state based on upper/lower Arc band intensity. (F) Arc immunoblot in dentate gyrus lysate input samples. β-Actin was used as a loading control. n = 4/5; Student’s t -test, * P < 0.05.

Article Snippet: Antibodies: Arc C7 mouse monoclonal (1:200, sc-17839), Arc H300 rabbit polyclonal (1:200, sc-15325), Cofilin (1:500, sc-32158), Drebrin A (1:200, #sc-374269), Dynamin 2 (1:1000, sc-6400), GAPDH (1:5000, sc-32233), Histone 1 (1:500, sc-10806), rabbit polyclonal SUMO1 (1:1000, sc-9060), mouse monoclonal SUMO1 (1:1000, sc-5308), SUMO2/3 (1:1000, sc-32873), normal mouse IgG and rabbit IgG were from Santa Cruz Biotechnology.

Techniques: In Vivo, Western Blot, Immunoprecipitation

Journal: Frontiers in Synaptic Neuroscience

Article Title: Dynamic Arc SUMOylation and Selective Interaction with F-Actin-Binding Protein Drebrin A in LTP Consolidation In Vivo

doi: 10.3389/fnsyn.2017.00008

Figure Lengend Snippet: 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.

Article Snippet: Antibodies: Arc C7 mouse monoclonal (1:200, sc-17839), Arc H300 rabbit polyclonal (1:200, sc-15325), Cofilin (1:500, sc-32158), Drebrin A (1:200, #sc-374269), Dynamin 2 (1:1000, sc-6400), GAPDH (1:5000, sc-32233), Histone 1 (1:500, sc-10806), rabbit polyclonal SUMO1 (1:1000, sc-9060), mouse monoclonal SUMO1 (1:1000, sc-5308), SUMO2/3 (1:1000, sc-32873), normal mouse IgG and rabbit IgG were from Santa Cruz Biotechnology.

Techniques: Modification, Protease Inhibitor, Lysis, Immunoprecipitation, Western Blot

Journal: Frontiers in Synaptic Neuroscience

Article Title: Dynamic Arc SUMOylation and Selective Interaction with F-Actin-Binding Protein Drebrin A in LTP Consolidation In Vivo

doi: 10.3389/fnsyn.2017.00008

Figure Lengend Snippet: Newly synthesized brain-derived neurotrophic factor (BDNF)-induced Arc is SUMOylated during LTP consolidation. (A) Time course plots of medial perforant path-dentate gyrus evoked fEPSPs recorded before and after HFS (indicated by arrows). Values are mean ± SEM of the maximum fEPSP slope expressed as percent of baseline. TrkB-Fc or control IgG-Fc (1 μl, 12.5 min, 100 μg) was infused into the dorsal dentate gyrus at 2 h post-HFS (indicated by a bar) and dentate gyrus tissue was collected at 4 h post-HFS (stippled line). TrkB-Fc infusion reverted ongoing LTP maintenance. (B) Bi-directional coimmunoprecipitation of SUMOylated Arc using anti-SUMO1 and anti-Arc antibodies. Enhanced Arc expression and Arc SUMO1ylation in HFS-treated dentate gyrus was reverted to baseline levels by TrkB-Fc infusion. (C) Bar graph shows fold change in Arc SUMOylation state in HFS-treated dentate gyrus compared to contralateral control in TrkB-Fc and IgG-Fc infused rats. Arc immunoprecipitation was performed and the ratio of 65 kDa (SUMO-Arc) to unmodified 50 kDa Arc was calculated. n = 5; Student’s t -test, * P < 0.05.

Article Snippet: Antibodies: Arc C7 mouse monoclonal (1:200, sc-17839), Arc H300 rabbit polyclonal (1:200, sc-15325), Cofilin (1:500, sc-32158), Drebrin A (1:200, #sc-374269), Dynamin 2 (1:1000, sc-6400), GAPDH (1:5000, sc-32233), Histone 1 (1:500, sc-10806), rabbit polyclonal SUMO1 (1:1000, sc-9060), mouse monoclonal SUMO1 (1:1000, sc-5308), SUMO2/3 (1:1000, sc-32873), normal mouse IgG and rabbit IgG were from Santa Cruz Biotechnology.

Techniques: Synthesized, Derivative Assay, Expressing, Immunoprecipitation

Journal: Frontiers in Synaptic Neuroscience

Article Title: Dynamic Arc SUMOylation and Selective Interaction with F-Actin-Binding Protein Drebrin A in LTP Consolidation In Vivo

doi: 10.3389/fnsyn.2017.00008

Figure Lengend Snippet: 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.

Article Snippet: Antibodies: Arc C7 mouse monoclonal (1:200, sc-17839), Arc H300 rabbit polyclonal (1:200, sc-15325), Cofilin (1:500, sc-32158), Drebrin A (1:200, #sc-374269), Dynamin 2 (1:1000, sc-6400), GAPDH (1:5000, sc-32233), Histone 1 (1:500, sc-10806), rabbit polyclonal SUMO1 (1:1000, sc-9060), mouse monoclonal SUMO1 (1:1000, sc-5308), SUMO2/3 (1:1000, sc-32873), normal mouse IgG and rabbit IgG were from Santa Cruz Biotechnology.

Techniques: In Vivo, Western Blot, Immunoprecipitation

Journal: Frontiers in Synaptic Neuroscience

Article Title: Dynamic Arc SUMOylation and Selective Interaction with F-Actin-Binding Protein Drebrin A in LTP Consolidation In Vivo

doi: 10.3389/fnsyn.2017.00008

Figure Lengend Snippet: Enhanced Arc SUMOylation in the cytoskeletal fraction during LTP in vivo . (A) Immunoblot characterization of dentate gyrus subcellular fractions. Ipsilateral HFS-treated (Ipsi), contralateral control (Contra), and Naïve. Vimentin and histone 1 were used as markers of the cytoskeletal and nuclear fractions, respectively. Tissue was collected 1 h post-HFS and fractions collected from four dentate gyri were pooled. A 65 kDa Arc immunoreactive band (indicated by an arrow) was detected in the cytoskeletal fraction. (B) Bidirectional co-immunoprecipitation using anti-SUMO1 and anti-Arc antibodies was performed in dentate gyrus cytoskeletal fractions. SUMO1ylated Arc was detected at 65 kDa. Representative blots based on three independent biological replicates. Sameples from two dentate gyrus were pooled. Note the absence of unmodified Arc in the SUMO1 pellet. (C) Arc immunoprecipitation was performed in the cytoskeletal fraction from HFS-treated and control dentate gyrus. (D) Arc immunoblot analysis of cytoskeletal fraction. Bar graphs shows increase in Arc expression and enhanced Arc SUMOylation based on the ratio of upper/lower Arc bands in the dentate gyrus cytoskeletal fraction at 1 h post-HFS relative to contralateral control. n = 5; Student’s t -test, * P < 0.05. Representative Arc immunoblots on right. Arrow indicates 65 kDa SUMOylated Arc.

Article Snippet: Antibodies: Arc C7 mouse monoclonal (1:200, sc-17839), Arc H300 rabbit polyclonal (1:200, sc-15325), Cofilin (1:500, sc-32158), Drebrin A (1:200, #sc-374269), Dynamin 2 (1:1000, sc-6400), GAPDH (1:5000, sc-32233), Histone 1 (1:500, sc-10806), rabbit polyclonal SUMO1 (1:1000, sc-9060), mouse monoclonal SUMO1 (1:1000, sc-5308), SUMO2/3 (1:1000, sc-32873), normal mouse IgG and rabbit IgG were from Santa Cruz Biotechnology.

Techniques: In Vivo, Western Blot, Immunoprecipitation, Expressing