mouse monoclonal anti sumo 1  (Developmental Studies Hybridoma Bank)

Journal: EMBO Reports

Article Title: SUMOylation of protein phosphatase 5 regulates phosphatase activity and substrate release

doi: 10.1038/s44319-024-00250-2

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Mouse anti-SUMO-1 (2A12) , Cell Signaling Technology , Cat# 5718; RRID:AB_10547145.

Techniques: Recombinant, Sequencing, Synthesized, Software, In Vitro

Journal: Arthritis Research & Therapy

Article Title: Decreased catalytic function with altered sumoylation of DNA topoisomerase I in the nuclei of scleroderma fibroblasts

doi: 10.1186/ar3435

Figure Lengend Snippet: Real-time RT-PCR and Western blots for SUMO1 with and without SUMO1 siRNA transfection in fibroblasts . Three SSc fibroblast strains (two with anti-topo I and one with anti-RNA polymerase III positive serum) were transfected with SUMO1 siRNA. After 48-hour transfection, total RNAs were used for measuring SUMO1 transcript levels (Figure 4a), and the nuclear extracts were used for measuring SUMO1 protein (Figure 4b). Error bars indicate standard deviation.

Article Snippet: Equal amounts of protein (500 ug) from each sample were subjected to immunoprecipitation (IP) with mouse anti-SUMO-1(GMP1, Invitrogen, Carlsbad, CA, USA) using nuclear complex co-IP kit (Active Motif, Carlsbad, CA), and then subjected to SDS-polyacrylamide gel electrophoresis.

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Standard Deviation

Journal: Arthritis Research & Therapy

Article Title: Decreased catalytic function with altered sumoylation of DNA topoisomerase I in the nuclei of scleroderma fibroblasts

doi: 10.1186/ar3435

Figure Lengend Snippet: Catalytic function of topo I in cultured SSc fibroblasts with and without SUMO1 siRNA transfection . A serial dilution of the nuclear extract containing topo I obtained from SSc fibroblasts was used to relax 0.25 μg supercoiled DNA. In this figure, the supercoiled DNA band was completely transformed to relaxed DNA at dilutions of one half and one in the fibroblasts without siRNA transfection or non-target siRNA transfection. In contrast, this change was observed between the one-eighth and one-fourth dilutions in the fibroblasts with SUMO1 transfection, which indicates a higher efficiency of catalytic function of topo I after SUMO1 inhibition in the fibroblasts. According to the intensity of the bands of remaining supercoiled DNA in serial dilutions in the assays of three fibroblast strains, these changes are significant. The P- values are 0.045 and 0.027 at the one-fourth dilution for comparisons between SUMO1 siRNA vs. non-target siRNA, or vs. without siRNA transfected fibroblasts, respectively (Student's t -test). This is representative of three SSc fibroblast strains examined in SUMO1 siRNA studies. * A , supercoiled DNA; B , relaxed DNA.

Article Snippet: Equal amounts of protein (500 ug) from each sample were subjected to immunoprecipitation (IP) with mouse anti-SUMO-1(GMP1, Invitrogen, Carlsbad, CA, USA) using nuclear complex co-IP kit (Active Motif, Carlsbad, CA), and then subjected to SDS-polyacrylamide gel electrophoresis.

Techniques: Cell Culture, Transfection, Serial Dilution, Transformation Assay, Inhibition

Journal: Arthritis Research & Therapy

Article Title: Decreased catalytic function with altered sumoylation of DNA topoisomerase I in the nuclei of scleroderma fibroblasts

doi: 10.1186/ar3435

Figure Lengend Snippet: Western blots show sumoylation of recombinant human topo I . Recombinant human topo I protein was subjected to the sumoylation reaction and examined by Western blotting using anti-topo I (I) and anti-SUMO1 antibodies (II). Compared to topo I protein without sumoylation reaction (topo I A ), topo I protein with sumoylation reaction (topo I B ) showed poly-sumoylation of topo I (II). The assays showed similar results in triplicates.

Article Snippet: Equal amounts of protein (500 ug) from each sample were subjected to immunoprecipitation (IP) with mouse anti-SUMO-1(GMP1, Invitrogen, Carlsbad, CA, USA) using nuclear complex co-IP kit (Active Motif, Carlsbad, CA), and then subjected to SDS-polyacrylamide gel electrophoresis.

Techniques: Western Blot, Recombinant

Journal: Arthritis Research & Therapy

Article Title: Decreased catalytic function with altered sumoylation of DNA topoisomerase I in the nuclei of scleroderma fibroblasts

doi: 10.1186/ar3435

Figure Lengend Snippet: Measurement of catalytic function of recombinant human topo I with and without sumoylation reaction . Recombinant human topo I proteins were sumoylated with either mutant sumo1 or wild type sumo1 or negative control (without sumoylation), and then were examined for their catalytic function in a serial dilution. Sumoylation of topo I with wild type sumo1 showed a reduction of efficiency in catalytic function (supercoiled DNA disappeared at the dilution of topo I concentration of 30) compared to the topo I protein sumoylated with mutant sumo1 or negative control (supercoiled DNA disappeared at topo I concentration of 22.5). This is representative of three assays. * A , standard supercoiled DNA band; B , standard relaxed DNA bands.

Article Snippet: Equal amounts of protein (500 ug) from each sample were subjected to immunoprecipitation (IP) with mouse anti-SUMO-1(GMP1, Invitrogen, Carlsbad, CA, USA) using nuclear complex co-IP kit (Active Motif, Carlsbad, CA), and then subjected to SDS-polyacrylamide gel electrophoresis.

Techniques: Recombinant, Mutagenesis, Negative Control, Serial Dilution, Concentration Assay

Journal: PLoS ONE

Article Title: Complex SUMO-1 Regulation of Cardiac Transcription Factor Nkx2-5

doi: 10.1371/journal.pone.0024812

Figure Lengend Snippet: (A) In situ hybridization analyses of SUMO pathway components at different developmental stages in the mouse (from 8.0 to 11.5 dpc, with ventral view at 8.0 dpc, and lateral view 9.5–11.5 dpc). Transcripts were seen in most embryonic regions, but enhanced expression could be noted in sites of extensive morphogenesis, such as the neural folds (8.0 dpc), branchial arches and limb buds (9.5 to 11.5 dpc). Arrows indicate cardiogenic regions at 8.0 dpc. (B) In situ hybridization revealed cardiac expression of SUMO-1 and SUMO-2, with stronger expression in the outer curvature, as demonstrated by Nppa transcripts. la – left atria; lv – left ventricle; rv – right ventricle; ra – right atria; oft – outflow tract.

Article Snippet: Immunohistochemistry was performed using rabbit polyclonal anti-Nkx2-5 antibody (1∶100 dilution, St. Cruz Biotechnology), mouse monoclonal anti-SUMO-1 (1∶100 Developmental Studies Hybridoma Bank), and anti-mouse IgG HRP-conjugated secondary antibody (1∶250 dilution, Amersham Biosciences), as previously described .

Techniques: In Situ Hybridization, Expressing

Journal: PLoS ONE

Article Title: Complex SUMO-1 Regulation of Cardiac Transcription Factor Nkx2-5

doi: 10.1371/journal.pone.0024812

Figure Lengend Snippet: (A) Transiently transfected HEK293T cells expressing Nkx2-5 and HA-SUMO-1 proteins alone or simultaneously. Addition of HA-SUMO-1 led to the appearance of two extra bands by western blot, specific for both HA and Nkx2-5 antibodies. (B) Co-IP experiments performed in cardiac HL-1 cells with Nkx2-5 antibody display a similar pattern of SUMOylation (arrowheads) but with increased detection of the slow-migrating SUMOylated Nkx2-5 band. Stars (*) indicate SUMOylated proteins co-precipitated with Nkx2-5 antibodies. (C) Incubation of cellular extracts with λPPA caused the disappearance of the slower migrating band detected by Nkx2-5 antibodies (lower left panel), while no change in the pattern of migration of the Nkx2-5/SUMO-1 co-stained bands was observed (upper left panel).

Article Snippet: Immunohistochemistry was performed using rabbit polyclonal anti-Nkx2-5 antibody (1∶100 dilution, St. Cruz Biotechnology), mouse monoclonal anti-SUMO-1 (1∶100 Developmental Studies Hybridoma Bank), and anti-mouse IgG HRP-conjugated secondary antibody (1∶250 dilution, Amersham Biosciences), as previously described .

Techniques: Transfection, Expressing, Western Blot, Co-Immunoprecipitation Assay, Incubation, Migration, Staining

Journal: PLoS ONE

Article Title: Complex SUMO-1 Regulation of Cardiac Transcription Factor Nkx2-5

doi: 10.1371/journal.pone.0024812

Figure Lengend Snippet: (A) Nkx2-5 sequences from fish to humans were analyzed bioinformatically and canonical conserved sites were identified at the amino-terminal region of the protein. (B–C) HEK293T cells were co-transfected with HA-SUMO-1 and single putative SUMOylation site mutants (K51R, K103R, K109R or the triple mutant 3K–R), and the band pattern was assessed by western blot with antibodies for HA and Nkx2-5. Transfection of K51R caused the disappearance of one previously detected SUMOylated band, indicating that K51 is a site for SUMO modification. All other mutants had identical SUMOylation pattern to the wild-type protein, suggesting that those were not SUMOylated sites. TND, TN domain; HD, homeodomain; NKD, NK2 specific domain; YRD, tyrosine-rich domain.

Article Snippet: Immunohistochemistry was performed using rabbit polyclonal anti-Nkx2-5 antibody (1∶100 dilution, St. Cruz Biotechnology), mouse monoclonal anti-SUMO-1 (1∶100 Developmental Studies Hybridoma Bank), and anti-mouse IgG HRP-conjugated secondary antibody (1∶250 dilution, Amersham Biosciences), as previously described .

Techniques: Transfection, Mutagenesis, Western Blot, Modification

Journal: PLoS ONE

Article Title: Complex SUMO-1 Regulation of Cardiac Transcription Factor Nkx2-5

doi: 10.1371/journal.pone.0024812

Figure Lengend Snippet: (A) HEK293T cells were transiently transfected with Nkx2-5, HA-SUMO-1 and the Nppa promoter. Activity is expressed as the fold-increase in luciferase expression. (B) No significant SUMO-1-mediated activation of the Nppa promoter was observed for other cardiac transcriptions factors (Tbx20a and GATA4/5), indicating that this effect was specific to Nkx2-5. (C) HEK293T were transiently transfected with wildtype Nkx2-5 or with a homeodomain point mutant that decreases DNA binding affinity. Addition of mutant protein abolishes SUMO-mediated activation. (D) The Drosophila homolog of Nkx2-5 gene, tinman , also displays SUMO-1-dependent transcriptional activity.

Article Snippet: Immunohistochemistry was performed using rabbit polyclonal anti-Nkx2-5 antibody (1∶100 dilution, St. Cruz Biotechnology), mouse monoclonal anti-SUMO-1 (1∶100 Developmental Studies Hybridoma Bank), and anti-mouse IgG HRP-conjugated secondary antibody (1∶250 dilution, Amersham Biosciences), as previously described .

Techniques: Transfection, Activity Assay, Luciferase, Expressing, Activation Assay, Mutagenesis, Binding Assay

Journal: PLoS ONE

Article Title: Complex SUMO-1 Regulation of Cardiac Transcription Factor Nkx2-5

doi: 10.1371/journal.pone.0024812

Figure Lengend Snippet: (A–C) SUMO-1 and Nkx2-5 failed to activate the cardiac promoters Gja5 (A) and Pitx2 (B) but could weakly activate Isl1 (B) and SM22 (C) promoters in HEK293T cells. (D) qPCR from First Heart Field (FHF) and trunk region enriched for Second Heart Field progenitors (eSHF) show presence of several SUMO components in both regions analysed. Regions used in this experimented are represented as red lines on E8.5 mouse embryo diagram (left). The relative levels on both regions were shown by non-saturated cycling PCR (30 cycles) followed by gel electrophoresis. (E) Immunohistochemistry of E9.5 embryos sections show Sumo-1 and Sumo-2 widely expressed and this pattern overlaps with Nkx2-5 expressing regions derived from Second Heart Field (SHF), including the outflow tract (oft) and lateral mesoderm (lm). lm – lateral mesoderm; oft – outflow tract; fg – foregut.

Article Snippet: Immunohistochemistry was performed using rabbit polyclonal anti-Nkx2-5 antibody (1∶100 dilution, St. Cruz Biotechnology), mouse monoclonal anti-SUMO-1 (1∶100 Developmental Studies Hybridoma Bank), and anti-mouse IgG HRP-conjugated secondary antibody (1∶250 dilution, Amersham Biosciences), as previously described .

Techniques: Nucleic Acid Electrophoresis, Immunohistochemistry, Expressing, Derivative Assay

Journal: PLoS ONE

Article Title: Complex SUMO-1 Regulation of Cardiac Transcription Factor Nkx2-5

doi: 10.1371/journal.pone.0024812

Figure Lengend Snippet: (A) SUMO-1 stimulates Nkx2-5-driven reporter activity in a dose-dependent manner, but SUMOylation-defective mutant proteins K51R and 3K-R had no effect on SUMO-dependent activation. Nkx2-5 and HA-SUMO1 protein levels were detected in cell extracts by western blot, with α-tubulin as loading control. (B) Immunofluorescence of transiently transfected HEK293T cells show normal nuclear localization of Nkx2-5 mutants K51R, K103R, K109R and 3K–R when compared to wildtype protein. (C) Nkx2-5 K51R binds to the NKE site with similar affinities to wildtype protein in HEK293T cells using EMSA. Overexpression of SUMO-1 leads to no detectable change in DNA affinities. S, specific oligonucleotide; NS, non-specific oligonucleotide; SS, supershift. Note that western blots were performed using extracts for Luciferase readings without NEM, therefore only the non-SUMOylated form of Nkx2-5 was detected.

Article Snippet: Immunohistochemistry was performed using rabbit polyclonal anti-Nkx2-5 antibody (1∶100 dilution, St. Cruz Biotechnology), mouse monoclonal anti-SUMO-1 (1∶100 Developmental Studies Hybridoma Bank), and anti-mouse IgG HRP-conjugated secondary antibody (1∶250 dilution, Amersham Biosciences), as previously described .

Techniques: Activity Assay, Mutagenesis, Activation Assay, Western Blot, Immunofluorescence, Transfection, Over Expression, Luciferase

Journal: PLoS ONE

Article Title: Complex SUMO-1 Regulation of Cardiac Transcription Factor Nkx2-5

doi: 10.1371/journal.pone.0024812

Figure Lengend Snippet: (A–B) Mutagenesis of every lysine in Nkx2-5 in conjunction with K51R failed to change its SUMOylation pattern, as detected by western blot. (B) Most double mutant proteins were not capable of attenuating the activation exerted by SUMO-1 on Nkx2-5 when the Nppa promoter was used in co-transfection experiments performed in HEK293T cells. The exception was the double mutant K51/191R mutant that showed strong activation. (C) Deletion of the carboxi-terminal region of Nkx2-5 (ΔC) impaired SUMO-mediated activation on the Nppa promoter, despite enhancing Nkx2-5 stability in transient assays (inset). (D) A SUMO-1/Nkx2-5 fusion construct failed to elicit any activation of the Nppa promoter when compared to the Nkx2-5 WT construct alone, but activation was restored upon addition of exogenous HA-SUMO-1 constructs. Inset: western blot of extracts used for luciferase readings show presence of exogenously expressed Nkx-2-5 WT, HA-SUMO-1 and SUMO-1/Nkx2-5 fusions (SUMO-1∼Nkx2-5) WT and K51R. Endogenous SUMO-1 and possibly RanGAP1 (*) are also detected.

Article Snippet: Immunohistochemistry was performed using rabbit polyclonal anti-Nkx2-5 antibody (1∶100 dilution, St. Cruz Biotechnology), mouse monoclonal anti-SUMO-1 (1∶100 Developmental Studies Hybridoma Bank), and anti-mouse IgG HRP-conjugated secondary antibody (1∶250 dilution, Amersham Biosciences), as previously described .

Techniques: Mutagenesis, Western Blot, Activation Assay, Cotransfection, Construct, Luciferase