35 s pfge patterns  (Bio-Rad)

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: In vitro translated [S 35 ]-labelled Nkx2-5 (TNT Quick Coupled System, Promega) or nuclear extracts from transfected HEK293T or HeLa cells were mixed with binding buffer (25 mM Hepes pH 7.4; 75 mM NaCl; 1 mM MgCl 2 ; 0.2 mM EDTA; 0.1% v/v NP-40; 1 mM DTT; 10 ng BSA; 200 ng poly dI/dC) and incubated for 10 min at room temperature.

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: In vitro translated [S 35 ]-labelled Nkx2-5 (TNT Quick Coupled System, Promega) or nuclear extracts from transfected HEK293T or HeLa cells were mixed with binding buffer (25 mM Hepes pH 7.4; 75 mM NaCl; 1 mM MgCl 2 ; 0.2 mM EDTA; 0.1% v/v NP-40; 1 mM DTT; 10 ng BSA; 200 ng poly dI/dC) and incubated for 10 min at room temperature.

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: In vitro translated [S 35 ]-labelled Nkx2-5 (TNT Quick Coupled System, Promega) or nuclear extracts from transfected HEK293T or HeLa cells were mixed with binding buffer (25 mM Hepes pH 7.4; 75 mM NaCl; 1 mM MgCl 2 ; 0.2 mM EDTA; 0.1% v/v NP-40; 1 mM DTT; 10 ng BSA; 200 ng poly dI/dC) and incubated for 10 min at room temperature.

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: In vitro translated [S 35 ]-labelled Nkx2-5 (TNT Quick Coupled System, Promega) or nuclear extracts from transfected HEK293T or HeLa cells were mixed with binding buffer (25 mM Hepes pH 7.4; 75 mM NaCl; 1 mM MgCl 2 ; 0.2 mM EDTA; 0.1% v/v NP-40; 1 mM DTT; 10 ng BSA; 200 ng poly dI/dC) and incubated for 10 min at room temperature.

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: In vitro translated [S 35 ]-labelled Nkx2-5 (TNT Quick Coupled System, Promega) or nuclear extracts from transfected HEK293T or HeLa cells were mixed with binding buffer (25 mM Hepes pH 7.4; 75 mM NaCl; 1 mM MgCl 2 ; 0.2 mM EDTA; 0.1% v/v NP-40; 1 mM DTT; 10 ng BSA; 200 ng poly dI/dC) and incubated for 10 min at room temperature.

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: In vitro translated [S 35 ]-labelled Nkx2-5 (TNT Quick Coupled System, Promega) or nuclear extracts from transfected HEK293T or HeLa cells were mixed with binding buffer (25 mM Hepes pH 7.4; 75 mM NaCl; 1 mM MgCl 2 ; 0.2 mM EDTA; 0.1% v/v NP-40; 1 mM DTT; 10 ng BSA; 200 ng poly dI/dC) and incubated for 10 min at room temperature.

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