mouse monoclonal anti sumo 1 antibody  (Thermo Fisher)

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

Journal: Comparative Cytogenetics

Article Title: Dual mechanism of chromatin remodeling in the common shrew sex trivalent (XY 1 Y 2 )

doi: 10.3897/CompCytogen.v11i4.13870

Figure Lengend Snippet: Mid-pachytene spermatocytes and male sex (XY 1 Y 2 ) chromosomes of Sorex araneus . Bar = 5µm. The axial elements of the SC and the kinetochores were localised using anti-SYCP3 ( green ) and anti-CREST ( red ) antibodies, respectively. a–e ATR ( magenta ) has a discontinuous localisation within the chromatin of the true sex chromosome regions (part of the X and the Y 1 ). The co-localisation of ATR , γH2AFX ( violet ), DAPI ( grey ) is shown in graph a - b (see c and c ’) f–j SUMO-1 ( yellow ) is localised on the chromatin of true sex chromosome regions. The co-localisation of SUMO-1 , γH2AFX ( violet ) and DAPI ( grey ) is shown in graph c - d (see h and h ’) k–o ubiH2A ( cyan ) is localised on the chromatin of the true sex chromosome regions. The co-localisation of ubiH2A , γH2AFX ( violet ) and DAPI ( grey ) is shown in graph e - f (see m and m ’) d, i, n Diagrams of the sex trivalents p, p’, p ’’ SYCP1 ( magenta ) is located on the area of chromosome synapsis of the autosomal part of the XY 1 Y 2 (from a-c ) q XY 1 Y 2 has two MLH1 signals ( yellow ). The MLH1 signal within the PAR synaptic site is marked by an asterisk. The arrowhead indicates the centromeres of the autosomal part of sex trivalent (part of the X and the Y 2 ) which are not co-oriented with each other ( red ).

Article Snippet: The slides were placed in phosphate buffer saline ( PBS ) and incubated overnight at 4°C with the primary antibodies diluted in antibody dilution buffer (3% bovine serum albumin - BSA, 0.05% Triton X-100 in PBS ): mouse anti- MLH1 (1:50–1:100, Abcam, Cambridge, UK), rabbit polyclonal anti-SYCP1 (1:500, Abcam, Cambridge, UK), rabbit polyclonal anti-SYCP3 (1:500–1:1000, Abcam, Cambridge, UK), mouse monoclonal anti- ATR (1:200, Abcam, Cambridge, UK), human anticentromere antibody CREST (Calcinosis Raynaud’s phenomenon, Esophageal dysmotility, Sclerodactyly, and Telangiectasia) (1:500, Fitzgerald Industries International, Acton, MA, USA), mouse monoclonal anti- SUMO-1 (1:250, Zymed Laboratories, South San Francisco, CA, USA), mouse monoclonal anti-ubiquityl histone H2A (1:400, Millipore, Billerica, MA, USA), and mouse anti-phospho-histone H2AX (also known as γH2AFX) (1:1000, Abcam, Cambridge, UK).

Techniques:

Journal: Comparative Cytogenetics

Article Title: Dual mechanism of chromatin remodeling in the common shrew sex trivalent (XY 1 Y 2 )

doi: 10.3897/CompCytogen.v11i4.13870

Figure Lengend Snippet: Mid-pachytene spermatocytes of Sorex araneus . Double immunostaining with antibodies: a–c anti-SYCP3 ( green )/anti- ubiH2A ( cyan ) d–f anti-SYCP3 ( green )/anti- SUMO-1 ( yellow ) g–i anti-SYCP3 ( green )/anti-RNA Pol II ( blue ) j–l anti-SYCP3 ( green )/anti-γH2AFX ( violet ). The true sex chromosome region is designated as XY 1 . Scale bars: 5 µm.

Article Snippet: The slides were placed in phosphate buffer saline ( PBS ) and incubated overnight at 4°C with the primary antibodies diluted in antibody dilution buffer (3% bovine serum albumin - BSA, 0.05% Triton X-100 in PBS ): mouse anti- MLH1 (1:50–1:100, Abcam, Cambridge, UK), rabbit polyclonal anti-SYCP1 (1:500, Abcam, Cambridge, UK), rabbit polyclonal anti-SYCP3 (1:500–1:1000, Abcam, Cambridge, UK), mouse monoclonal anti- ATR (1:200, Abcam, Cambridge, UK), human anticentromere antibody CREST (Calcinosis Raynaud’s phenomenon, Esophageal dysmotility, Sclerodactyly, and Telangiectasia) (1:500, Fitzgerald Industries International, Acton, MA, USA), mouse monoclonal anti- SUMO-1 (1:250, Zymed Laboratories, South San Francisco, CA, USA), mouse monoclonal anti-ubiquityl histone H2A (1:400, Millipore, Billerica, MA, USA), and mouse anti-phospho-histone H2AX (also known as γH2AFX) (1:1000, Abcam, Cambridge, UK).

Techniques: Double Immunostaining

Journal: Comparative Cytogenetics

Article Title: Dual mechanism of chromatin remodeling in the common shrew sex trivalent (XY 1 Y 2 )

doi: 10.3897/CompCytogen.v11i4.13870

Figure Lengend Snippet: Intensity correlation analysis ( ICA ) represented by scatter plots showing the paired intensities of two channels ( a γH2AFX - ATR , Fig. b γH2AFX - SUMO-1 , Fig. c γH2AFX - ubiH2A Fig. ). r p - Pearson correlation coefficient. See more details in the text. Degree of co-localisation for signals in sex trivalents of common shrew ( d ). On the y -axis, the percentage of co-localised signals are shown according to overlap correlation coefficients ( r ) and the Pearson correlation coefficient ( r p ).

Article Snippet: The slides were placed in phosphate buffer saline ( PBS ) and incubated overnight at 4°C with the primary antibodies diluted in antibody dilution buffer (3% bovine serum albumin - BSA, 0.05% Triton X-100 in PBS ): mouse anti- MLH1 (1:50–1:100, Abcam, Cambridge, UK), rabbit polyclonal anti-SYCP1 (1:500, Abcam, Cambridge, UK), rabbit polyclonal anti-SYCP3 (1:500–1:1000, Abcam, Cambridge, UK), mouse monoclonal anti- ATR (1:200, Abcam, Cambridge, UK), human anticentromere antibody CREST (Calcinosis Raynaud’s phenomenon, Esophageal dysmotility, Sclerodactyly, and Telangiectasia) (1:500, Fitzgerald Industries International, Acton, MA, USA), mouse monoclonal anti- SUMO-1 (1:250, Zymed Laboratories, South San Francisco, CA, USA), mouse monoclonal anti-ubiquityl histone H2A (1:400, Millipore, Billerica, MA, USA), and mouse anti-phospho-histone H2AX (also known as γH2AFX) (1:1000, Abcam, Cambridge, UK).

Techniques:

Journal: Comparative Cytogenetics

Article Title: Dual mechanism of chromatin remodeling in the common shrew sex trivalent (XY 1 Y 2 )

doi: 10.3897/CompCytogen.v11i4.13870

Figure Lengend Snippet: Schematic illustration of male common shrew MSCI . A mid-pachytene spermatocyte ( a ) and a sex (XY 1 Y 2 ) trivalent ( b ) of a shrew are shown. An electron micrograph of the sex trivalent is shown at the top of the b . The true sex chromosome regions (part of the X and the Y 1 ) form a sex body on the periphery of the nucleus. The chromatin of the sex body undergoes reorganisation. MSCI markers have different distributions: SUMO-1 ( yellow ), ATR ( black dots ), ubiH2A ( blue ), γH2AFX ( violet ). ATR is localised on the true sex chromosome regions, and is especially intense on the asynaptic region with a smaller amount where there is synapsis. SUMO-1 and ubiH2A are localised on both the asynaptic and synaptic regions of the true sex chromosome regions. γH2AFX overlays all the true sex chromosome regions and the unpaired part of the Y 2 axial element. Representative autosomal SCs are shown. MLH1 signals are shown as black balls. The red balls indicate centromeres.

Article Snippet: The slides were placed in phosphate buffer saline ( PBS ) and incubated overnight at 4°C with the primary antibodies diluted in antibody dilution buffer (3% bovine serum albumin - BSA, 0.05% Triton X-100 in PBS ): mouse anti- MLH1 (1:50–1:100, Abcam, Cambridge, UK), rabbit polyclonal anti-SYCP1 (1:500, Abcam, Cambridge, UK), rabbit polyclonal anti-SYCP3 (1:500–1:1000, Abcam, Cambridge, UK), mouse monoclonal anti- ATR (1:200, Abcam, Cambridge, UK), human anticentromere antibody CREST (Calcinosis Raynaud’s phenomenon, Esophageal dysmotility, Sclerodactyly, and Telangiectasia) (1:500, Fitzgerald Industries International, Acton, MA, USA), mouse monoclonal anti- SUMO-1 (1:250, Zymed Laboratories, South San Francisco, CA, USA), mouse monoclonal anti-ubiquityl histone H2A (1:400, Millipore, Billerica, MA, USA), and mouse anti-phospho-histone H2AX (also known as γH2AFX) (1:1000, Abcam, Cambridge, UK).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Trophic factor BDNF inhibits GABAergic signaling by facilitating dendritic enrichment of SUMO E3 ligase PIAS3 and altering gephyrin scaffold

doi: 10.1016/j.jbc.2022.101840

Figure Lengend Snippet: BDNF alters subcellular localization of SUMO pathway proteins. A–B , endogenous SUMO-1 and SUMO-2/3 staining in control neurons. A′-B′ , endogenous SUMO-1 and SUMO-2/3 staining in BDNF-treated neurons. Quantification of hippocampal neurons exhibiting dendritic/nuclear enrichment of SUMO-1 or SUMO-2/3. C , neurons transfected with myc-PIAS-1, myc-PIAS-2α, myc-PIAS-3, or myc-PIAS-γ in control and BDNF-treated neurons. D-D′ , neurons transfected with myc-PIAS-3 and treated with BDNF (90 min or 48 h). D’’ , neurons transfected with myc-PIAS-3 and treated with ANA-12 prior to BDNF application. Quantification of nuclear/dendritic enrichment of myc-PIAS-3. E-E′ , endogenous PIAS-3 showing nuclear localization in control neurons and dendritic enrichment after BDNF application. E’’ , endogenous PIAS-3 showing nuclear enrichment after TrkB-Fc coapplication with BDNF. Quantification of nuclear/dendritic enrichment of endogenous PIAS-3 after acute BDNF application or TrkB-Fc co-application. Three independent experiments N = 30, Scale bar 5 μm. BDNF, brain-derived neurotrophic factor; PIAS, protein inhibitor of activated STAT; SUMO, small ubiquitin-like modifier; TrkB, tropomyosin-related kinase B.

Article Snippet: Mouse anti-Gephyrin (1:1000, clones mAb7a, Synaptic Systems #147021), rabbit anti-SUMO-1 (1:250, Epitomics#1563-1), mouse anti-SUMO-1 (1:100, SantaCruz#sc-5308), rabbit anti-SUMO-2/3 (1:250, Cell signaling #4974), rabbit anti-SUMO-2/3 (1:250, Epitomics #2970-1), mouse anti-PIAS-3 (1:500, Sigma #P0117), rabbit anti-vGAT (1:2000, Synaptic Systems #131011); mouse anti-Myc tag (1:5000,Roche #11667149001), rabbit anti-Myc tag (1:5000, Cell Signaling #2278S), and mouse anti-FLAG tag (1:5000, Sigma Aldrich #F3165).

Techniques: Staining, Transfection, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: CCAAT/Enhancer-Binding Protein ε 27 Antagonism of GATA-1 Transcriptional Activity in the Eosinophil Is Mediated by a Unique N-Terminal Repression Domain, Is Independent of Sumoylation and Does Not Require DNA Binding

doi: 10.3390/ijms222312689

Figure Lengend Snippet: ( A , B ) C/EBPε is sumoylated in the eosinophil lineage. Detection of endogenous sumoylated C/EBPε in AML14.3D10 eosinophil lysates prepared in the presence of 50 μM N -ethylmaleimide, immunoprecipitated with anti-C/EBPε antibody, and detected by Western blotting using an anti-SUMO-1 antibody ( A ). Detection of sumoylated C/EBPε 32 and C/EBPε 27 in lysates of COS-7 cells transfected as indicated with expression vectors for C/EBPε 27 , C/EBPε 32 , FLAG-SUMO-1, and mutant-FLAG-SUMO-1 (a form of SUMO-1 in which the target glycine has been converted to alanine and is unavailable for conjugation) ( B ). Lysates prepared with 50 μM N -ethylmaleimide were immunoprecipitated with anti-C/EBPε antibody and the Western blot probed with anti-FLAG M2 HRP-conjugated antibody. ( C – E ) Over-expression of SUMO-1 or mutation of the C/EBPε 27 sumoylation site has no effect on the repressor activity of C/EBPε 27 for GATA-1 transactivation of the MBP1-P2 promoter. Co-transfection of a SUMO-1 expression vector with C/EBPε 32 or C/EBPε 27 , does not alter the repressor activity of C/EBPε 27 for GATA-1 trans-activation of the MBP1-P2 promoter, nor convert C/EBPε 32 into a repressor independently or for GATA-1 ( C ) (* p < 0.05, ***, ### p < 0.001, compared to GATA-1 alone, ±SUMO-1, respectively). Mutation of the sumoylation target lysine (K) residue in the conserved SUMO (VKEEP) consensus site to either arginine (K → R) or alanine (K → A) ( D ) does not alter the repressor activity of C/EBPε 27 for GATA-1 transactivation of the MBP1-P2 promoter ( E ) (** p < 0.01; ns , not significant, compared to GATA-1 alone). Mutation of this target lysine in full length C/EBPε 32 ( D ) does not convert it into an activator or repressor in the presence of GATA-1 ( E ).

Article Snippet: For detection of SUMO-1, either an anti-FLAG-HRP antibody (M2, Sigma Aldrich, St. louis, MI, USA) or a mouse anti-SUMO-1 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA) was used.

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing, Mutagenesis, Conjugation Assay, Over Expression, Activity Assay, Cotransfection, Plasmid Preparation, Activation Assay

Journal: Journal of Molecular Cell Biology

Article Title: SUMOylation of α-tubulin is a novel modification regulating microtubule dynamics

doi: 10.1093/jmcb/mjaa076

Figure Lengend Snippet: α-tubulin is SUMOylated in cells and in vitro . ( A ) Immunoprecipitates with α-tubulin Ab from HEK293 cells expressing Flag-SUMO1, Flag-SUMO2, or Flag-SUMO3 were subject to immunoblotting (IB) and probed with Flag and α-tubulin (α-Tub) Abs. ( B ) Endogenous α-tubulin in HEK293 cells was immunoprecipitated and probed with SUMO1 Ab. ( C ) Immunoprecipitates with α-tubulin Ab from HEK293 cells expressing HA-Ubc9 were probed with HA and α-tubulin Abs. ( D ) Coomassie blue staining of purified mouse brain tubulin including α-tubulin and β-tubulin, indicated by arrows. ( E ) Coomassie blue staining of purified GST-SAE2/1, GST-Ubc9, His-SUMO1GG, and His-SUMO1ΔGG. Asterisk indicates the band of purified protein. ( F ) In vitro SUMOylation assay using purified GST-SAE2/1, GST-Ubc9, His-SUMO1GG, and brain tubulins. ( G ) Ratio of density of SUMOylated bands to unSUMOylated bands. ( H ) Purified tubulin was in vitro SUMOylated and probed with α-tubulin Ab. ( I ) In vitro SUMOylation assay using soluble tubulins and MTs. ( J ) Immunoprecipitates with β-tubulin Ab from HEK293 cells expressing Flag-SUMO1, Flag-SUMO2, or Flag-SUMO3 were probed with Flag and β-tubulin Abs. ( K ) Purified tubulin was in vitro SUMOylated and probed with β-tubulin Ab. ( L ) Endogenous β-tubulin in HEK293 cells was immunoprecipitated and probed with SUMO1 Ab. The experiments were repeated three times.

Article Snippet: The primary antibodies included mouse antibodies against SUMO1 (1:1000; CST, 4940S), SUMO2/3 (1:1000; CST, 4971S), α-tubulin (1:10000; Sigma, T9026), α-tubulin (1:10000; Abcam, ab18251), Flag (1:10000; Sigma, F7425), β-actin (1:50000; Chemicon, MAB1501), and GAPDH (1:10000; Abcam, ab8245).

Techniques: In Vitro, Expressing, Western Blot, Immunoprecipitation, Staining, Purification

Journal: Journal of Molecular Cell Biology

Article Title: SUMOylation of α-tubulin is a novel modification regulating microtubule dynamics

doi: 10.1093/jmcb/mjaa076

Figure Lengend Snippet: SUMOylation is mainly enriched in soluble α-tubulin. ( A ) PLA with α-tubulin and SUMO1 Abs was performed in HEK293 cells. Confocal images of PLA signals and tubulin labelled after PLA are shown. The enlarged image of the boxed area is shown at the lower right. Scale bar, 10 μm. ( B ) PLA dots on and off MTs were quantified. n = 11 cells. ( C ) Soluble and polymerized tubulins were separated in SUMO1-overexpressing HEK293 cells and subject to IP using α-tubulin Ab. Immunoprecipitates were detected by SUMO1 Ab. The experiments were repeated three times. ( D ) Quantification of the SUMOylation in soluble or polymerized tubulins. Data are mean ± SEM from three independent experiments.

Article Snippet: The primary antibodies included mouse antibodies against SUMO1 (1:1000; CST, 4940S), SUMO2/3 (1:1000; CST, 4971S), α-tubulin (1:10000; Sigma, T9026), α-tubulin (1:10000; Abcam, ab18251), Flag (1:10000; Sigma, F7425), β-actin (1:50000; Chemicon, MAB1501), and GAPDH (1:10000; Abcam, ab8245).

Techniques:

Journal: Journal of Molecular Cell Biology

Article Title: SUMOylation of α-tubulin is a novel modification regulating microtubule dynamics

doi: 10.1093/jmcb/mjaa076

Figure Lengend Snippet: α-tubulin is SUMOylated at K96, K166, and K304 and deSUMOylated by SENP1. ( A ) Alignment of lysines (in red color) and surrounding sequences of α-tubulin isotypes in the mouse. ( B ) Immunoprecipitates with Flag M2 beads from HEK293T cells expressing HA-SUMO1 with Flag-tagged isotypes of α-tubulin as indicated were probed with SUMO1 and Flag Abs. ( C ) Flag-tagged wild-type (WT) and various α1A mutant at MS-predicted SUMOylation sites were expressed with HA-SUMO1 in CHO-K1 cells and then immunoprecipitated. Immunoprecipitates were detected by SUMO1 and Flag Abs. ( D ) List of putative SUMOylation sites on α1A isotype predicted by different softwares. ( E ) Flag-tagged WT and combined mutant α1A at bioinformatics-predicted SUMOylation sites were expressed with HA-SUMO1 in CHO-K1 cells and then immunoprecipitated. Immunoprecipitates were detected by SUMO1 and Flag Abs. ( F ) Schematic representation of SUMOylation sites on the structure of α-tubulin. K96, K166, and K304 are shown as red spheres. ( G ) Location of SUMOylation sites and the interface for lateral contact between two α-tubulins in one MT. K96, K166, and K304 are shown as red spheres, and H2-S3 loop and M-loop are in blue. ( H ) Flag-α1A was overexpressed with HA-SUMO1 or HA-SUMO1-K7,16,17R mutant in HEK293 cells. Immunoprecipitates using Flag M2 beads were probed with HA and Flag Abs. ( I ) Immunoprecipitates with Flag M2 beads from HEK293 cells expressing Flag-SENP1 or Flag-SENP2 were probed with Flag and α-tubulin Abs. ( J ) Immunoprecipitates with α-tubulin from HEK293 cells expressing Flag-SENP1 or Flag-SENP2 with Flag-SUMO1 were probed with SUMO1 and α-tubulin Abs. ( K ) Immunoprecipitates from E13.5 brain of SENP1 +/+ and SENP1 –/– mice were probed with SUMO1 and α-tubulin Abs. The experiments were repeated three times.

Article Snippet: The primary antibodies included mouse antibodies against SUMO1 (1:1000; CST, 4940S), SUMO2/3 (1:1000; CST, 4971S), α-tubulin (1:10000; Sigma, T9026), α-tubulin (1:10000; Abcam, ab18251), Flag (1:10000; Sigma, F7425), β-actin (1:50000; Chemicon, MAB1501), and GAPDH (1:10000; Abcam, ab8245).

Techniques: Expressing, Mutagenesis, Immunoprecipitation

Journal: European Journal of Pain (London, England)

Article Title: Alterations in SUMOylation of the hyperpolarization‐activated cyclic nucleotide‐gated ion channel 2 during persistent inflammation

doi: 10.1002/ejp.1606

Figure Lengend Snippet: HCN2 is SUMOylated in rat DRG. Denaturing immunoprecipitation (IP) experiments were performed on a DRG membrane preparation with an antibody against HCN2 or IgG (control). IP products from 1 membrane preparation were run in triplicate on an SDS‐polyacrylamide gel followed by western blotting (WB). The blot was cut into 3, and probed for HCN2, SUMO2/3 and SUMO1. The experiment was repeated using three different DRG membrane preparations. A representative result from one experiment is shown. All three WB antibodies recognized the same ~100 kD protein in the HCN2 IP product but not the IgG IP product. The data indicate that SUMO1 and SUMO2/3 are covalently linked to HCN2 since they remained bound under denaturing conditions. The ~50 kD band present in all lanes represents the IP antibody. DRG, dorsal root ganglia; HCN2, hyperpolarization‐activated, cyclic nucleotide‐gated 2; SUMO, s mall u biquitin like mo difier

Article Snippet: For PLA, a monoclonal mouse antibody against SUMO1 (Santa Cruz, Sc‐5308) was used at 1:100, and a monoclonal mouse antibody against SUMO2/3 (Developmental Studies Hybridoma Bank, SUMO‐2 8A2) was used at a concentration of 1:70 and developed by Matinus, M. at John Hopkins School of Medicine, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology.

Techniques: Immunoprecipitation, Western Blot

Journal: European Journal of Pain (London, England)

Article Title: Alterations in SUMOylation of the hyperpolarization‐activated cyclic nucleotide‐gated ion channel 2 during persistent inflammation

doi: 10.1002/ejp.1606

Figure Lengend Snippet: Measuring HCN2 channel SUMOylation. (a) Verification of SUMO antibodies. Antibodies were (right) or were not (left) preabsorbed with the corresponding peptide for SUMO2/3 (upper panel) or SUMO1 (lower panel). SUMO is predominately expressed in nuclei. Note the loss of nuclear staining following preabsorption, for example, white arrows in bottom panel. Scale bars are 100 µm. (b) Method for quantifying HCN2 channel SUMOylation. In situ PLA was performed on DRG cryosections with (experimental) or without (control) antibodies against SUMO2/3 and HCN2. Upper panel shows a 5 µm projection of confocal optical slices through representative cells from control and experimental treatment groups. Each image represents a projection of five slices in continuous succession that together encompass the centre of the cell. Each optical section is 0.9 µm with a z‐stack interval of 1 µm. Note that the red puncta indicating SUMOylated HCN2 channels were largely absent when antibodies were omitted. Scale bar is 10 µm. The cells were circle and thresholded, and the resulting image is shown in the lower panel. Black puncta within the circled region were counted using the analyse particle tool on imageJ and normalized by the area (µm 2 ) of the circle. Note that all black puncta, regardless of size were counted. (c) HCN2 channels are SUMOylated in DRG neurons. Plots of puncta per µm 2 show there are significantly more puncta when primary antibodies for HCN2 and SUMO2/3 were included (0.08 ± 0.01 vs. 0.16 ± 0.02, p = 0.0149, paired t ‐test, n = 3, 70 and 62 cells analysed in total) *, p < 0.05. The lines indicate that the cells were from the same experiment, that is, alternate sections from a single DRG on the same slide receiving the same PLA reagents and treated in an identical fashion. DRG, dorsal root ganglia; HCN2, hyperpolarization‐activated, cyclic nucleotide‐gated 2; PLA, proximity ligation assays; SUMO, s mall u biquitin like mo difier

Article Snippet: For PLA, a monoclonal mouse antibody against SUMO1 (Santa Cruz, Sc‐5308) was used at 1:100, and a monoclonal mouse antibody against SUMO2/3 (Developmental Studies Hybridoma Bank, SUMO‐2 8A2) was used at a concentration of 1:70 and developed by Matinus, M. at John Hopkins School of Medicine, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology.

Techniques: Staining, In Situ, Ligation

Journal: European Journal of Pain (London, England)

Article Title: Alterations in SUMOylation of the hyperpolarization‐activated cyclic nucleotide‐gated ion channel 2 during persistent inflammation

doi: 10.1002/ejp.1606

Figure Lengend Snippet: HCN2 channel SUMOylation by SUMO1 is diminished in medium and large diameter neurons from ipsilateral relative to contralateral DRG at 1 day post‐CFA. (a) Representative PLA. Scale bars are 25 µm. Note that this image provides an overview, but for the purpose of quantification, individual projections were made for each cell, for example, Figure . (b) Plots of mean number of puncta/µm 2 for all DRG from six CFA treated animals on day 1 post‐CFA The number inside the bar represents the total number of cells analysed. Inset: Each line compares means for contralateral and ipsilateral DRG from one animal. Paired t ‐tests indicate medium and large diameter cells contain significantly fewer puncta in the ipsilateral DRG relative to the contralateral DRG (small, 0.33 ± 0.05 vs. 0.27 ± 0.03, p = 0.2058; medium, 0.34 ± 0.05 vs. 0.24 ± 0.03, p = 0.0480; large, 0.37 ± 0.05 vs. 0.26 ± 0.02, p = 0.0238), * p < 0.05. (c) Plots of mean puncta intensity on day 1 post‐CFA. Paired t ‐tests indicate mean puncta intensities were not significantly different for any size category in ipsilateral compared to contralateral DRG (small, 44.68 ± 4.03 vs. 41.48 ± 2.66, p = 0.5986; medium, 42.7 ± 3.24 vs. 45.65 ± 4.06, p = 0.6299; large, 44.44 ± 3.68 vs. 46.39 ± 4.27, p = 0.7631). CFA, Complete Freund's Adjuvant; DRG, dorsal root ganglia; HCN2, hyperpolarization‐activated, cyclic nucleotide‐gated 2; PLA, proximity ligation assays; SUMO, s mall u biquitin like mo difier

Article Snippet: For PLA, a monoclonal mouse antibody against SUMO1 (Santa Cruz, Sc‐5308) was used at 1:100, and a monoclonal mouse antibody against SUMO2/3 (Developmental Studies Hybridoma Bank, SUMO‐2 8A2) was used at a concentration of 1:70 and developed by Matinus, M. at John Hopkins School of Medicine, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology.

Techniques: Ligation

Journal: European Journal of Pain (London, England)

Article Title: Alterations in SUMOylation of the hyperpolarization‐activated cyclic nucleotide‐gated ion channel 2 during persistent inflammation

doi: 10.1002/ejp.1606

Figure Lengend Snippet: HCN2 channel SUMOylation by SUMO1 is increased in small neurons from ipsilateral relative to contralateral DRG at 3 days post‐CFA. (a) Representative PLA. Scale bars are 25 µm. Note that this image provides an overview, but for the purpose of quantification, individual projections were made for each cell, for example, Figure . (b) Plots of mean puncta/µm 2 for all DRG from six CFA treated animals on day 3 post‐CFA. The number inside the bar represents the total number of cells analysed. Inset: Each line compares means for contralateral and ipsilateral DRG from one animal. Significantly more puncta were observed in ipsilateral relative to contralateral DRG as indicated by paired t ‐tests or non‐parametric alternative** (small, 0.25 ± 0.03 vs. 0.32 ± 0.03, p = 0.0285; medium, 0.29 ± 0.05 vs. 0.25 ± 0.06, p = 0.4310; large**, 0.32 ± 0.03 vs. 0.28 ± 0.06, p = 0.5625). *, p < 0.05 (c) Plots of mean puncta intensity on day 3 post‐CFA. Paired t ‐tests indicate mean puncta intensities were not significantly different for any size category in ipsilateral compared to contralateral DRG (small, 46.14 ± 3.0 vs. 46.08 ± 1.41, p = 0.9826; medium, 44.58 ± 4.12 vs. 46.45 ± 1.32, p = 0.6612; large, 44.99 ± 3.38 vs. 48.35 ± 1.27 p = 0.2758). CFA, Complete Freund's Adjuvant; DRG, dorsal root ganglia; HCN2, hyperpolarization‐activated, cyclic nucleotide‐gated 2; PLA, proximity ligation assays; SUMO, s mall u biquitin like mo difier

Article Snippet: For PLA, a monoclonal mouse antibody against SUMO1 (Santa Cruz, Sc‐5308) was used at 1:100, and a monoclonal mouse antibody against SUMO2/3 (Developmental Studies Hybridoma Bank, SUMO‐2 8A2) was used at a concentration of 1:70 and developed by Matinus, M. at John Hopkins School of Medicine, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology.

Techniques: Ligation