mouse antibodies targeting erg  (Santa Cruz Biotechnology)

Journal: Nature Communications

Article Title: The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature

doi: 10.1038/s41467-019-12897-w

Figure Lengend Snippet: ERG controls the expression of multiple regulators of coagulation in a tissue-specific manner. a qPCR screen analysis of genes involved in the regulation of coagulation in HUVEC was performed at 48 h after ERG inhibition by siRNA ( n = 4–6 independent experiments). Data were normalised to GAPDH . b , c qPCR analysis of selected targets genes directly or indirectly involved in coagulation and endothelial markers in whole liver ( b ) and lung ( c ) lysates from adult control ( Erg fl/fl ) and ERG-deficient ( Erg iEC-KO ) mice ( n = 4–5 per genotype). Data were normalised to 18S . d – e qPCR analysis of thrombomodulin ( TM) mRNA expression in whole liver ( d ) and lung ( e ) lysates from adult Erg fl/fl and Erg iEC-KO mice ( n = 5 per genotype). Data were normalised to 18S. f Immunoblot and quantification of WB for TM in protein extracts of lung samples from adult Erg fl/fl and Erg iEC-KO mice 30 days after tamoxifen injection ( n = 3 mice per group). Data were normalized to GAPDH. All graphical data are mean ± s.e.m, * P < 0.05, Student’s t -test. Source data are provided as a Source Data file

Article Snippet: ERG (mouse) , Santa Cruz , sc-376293 , PLA (HUVEC, 1/200)/WB (HUVEC, 1/2000).

Techniques: Expressing, Coagulation, Inhibition, Control, Western Blot, Injection

Journal: Nature Communications

Article Title: The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature

doi: 10.1038/s41467-019-12897-w

Figure Lengend Snippet: ERG regulates thrombomodulin expression and activity in vitro. a qPCR analysis of thrombomodulin ( TM ) mRNA expression in control (siCtrl) and ERG-deficient (siERG) HUVEC after 12, 24 and 48 h treatment ( n = 3 independent experiments). Data were normalized to GAPDH . b Representative immunoblot and quantification of TM following siCtrl or siERG treatment on HUVEC for 12, 24 and 48 h ( n = 4 independent experiments). Data were normalized to GAPDH. c Representative image and quantification of TM expression (green) in HUVEC transfected with siCtrl or siERG siRNA for 48 h by immunofluorescence; nuclei are identified by DAPI (blue) and cells are co- stained for ERG (magenta). Scale bar 40 µm. Quantification represents the mean pixel intensity for TM signal (arbitrary unit, A.U.) per cell. d , e Representative immunoblot and quantification of ERG and TM expression in control (siCtrl) and ERG-deficient (siERG) d HDMEC (microvascular EC) or e HDBEC (microvascular EC) after 48 h siRNA treatment ( n = 3). f qPCR analysis of TM mRNA expression in HUVEC transfected with control pcDNA or ERG cDNA expression plasmid (ERG) ( n = 3). Data were normalised to GAPDH . g siCtrl or siERG-treated HUVEC for 48 h were incubated with protein C (300 nM), CaCl 2 (5 mM), thrombin (10 nM). After 10, 20, 30 and 60 min of incubation at 37 °C, anti-thrombin III (100 nM) and heparin (15 U per ml) were added to neutralise thrombin, and protein C activity was measured using chromogenic substrate S-2366 (0.5 mM) ( n = 3). Data are expressed as relative activated protein C (APC) concentration normalised to siCtrl-treated condition following 60 min of incubation. All graphical data are mean ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t -test. Source data are provided as a Source Data file

Article Snippet: ERG (mouse) , Santa Cruz , sc-376293 , PLA (HUVEC, 1/200)/WB (HUVEC, 1/2000).

Techniques: Expressing, Activity Assay, In Vitro, Control, Western Blot, Transfection, Immunofluorescence, Staining, Plasmid Preparation, Incubation, Concentration Assay

Journal: Nature Communications

Article Title: The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature

doi: 10.1038/s41467-019-12897-w

Figure Lengend Snippet: ERG binds to and transactivates the thrombomodulin promoter in vitro. a Putative ERG binding sites (black bars) are located within the TM promoter upstream and downstream of the transcription start site (black arrow); ERG ChIP-sequencing data show a significant ERG peak on TM proximal promoter. ENCODE sequence conservation between 100 vertebrates is shown across this gene. ENCODE ChIP-seq data profiles for H3K4Me3 (tri-methylation of lysine (K) 4 on histone 3), H3K27Ac (acetylation of lysine (K) 27 on histone 3) and RNA polymerase II (RNA pol II) in HUVEC indicate open chromatin and active transcription. Location of qPCR primers covering regions R1 and R2 (black bar) are indicated. b ChIP-qPCR using primers to region R1 and R2 on ERG-bound chromatin from siCtrl or siERG-treated HUVEC. Primers for a region within 5′UTR region of TM gene (TM neg con) were used as negative control. Data are shown as fold change over IgG ( n = 3 independent experiments). Graphical data are mean ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t -test. c TM promoter luciferase reporter assay. ERG cDNA expression plasmid (ERG) or empty expression plasmid (pcDNA) were co-transfected with TM promoter-luciferase constructs (TM wild type (WT), TM mutant 1 or TM mutant 2) or a pGL4 empty vector in HUVEC, and luciferase activity was measured. Values represent the fold change in relative luciferase activity over the empty pGL4 vector alone ( n = 4 independent experiments). Graphical data are mean ± s.e.m., ** P < 0.01, *** P < 0.001, One-way ANOVA; ## P < 0.01 compared to pcDNA-TM WT condition, Student’s t -test. d ChIP-qPCR using primers to region R1 and R2 on H3K27Ac-bound chromatin from HUVEC treated with DMSO or p300 inhibitor (10 µM) for 1 h. Data are shown as fold change over IgG ( n = 3 independent experiments). e – f ChIP-qPCR using primers to region R1 and R2 on e p300- ( n = 3 independent experiments) or f H3K27Ac-bound ( n = 4 independent experiments) chromatin from siCtrl or siERG-treated HUVEC. Data are shown as fold change over IgG. All graphical data for ChIP experiments are mean ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t -test. Source data are provided as a Source Data file

Article Snippet: ERG (mouse) , Santa Cruz , sc-376293 , PLA (HUVEC, 1/200)/WB (HUVEC, 1/2000).

Techniques: In Vitro, Binding Assay, ChIP-sequencing, Sequencing, Methylation, ChIP-qPCR, Negative Control, Luciferase, Reporter Assay, Expressing, Plasmid Preparation, Transfection, Construct, Mutagenesis, Activity Assay

Journal: Nature Communications

Article Title: The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature

doi: 10.1038/s41467-019-12897-w

Figure Lengend Snippet: ERG cooperates with KLF2 to drive thrombomodulin expression. a – d TM promoter luciferase reporter assays. Values represent the fold change in relative luciferase activity over the empty pGL4 vector alone. b pcDNA or ERG expression plasmid (ERG) were co-transfected with a TM wild type (WT) promoter-luciferase construct (TM WT) in siCtrl or siKLF2-treated HUVEC, and luciferase activity was measured ( n = 3 independent experiments). b pcDNA or KLF2 cDNA expression plasmid (KLF2) were co-transfected with TM WT promoter construct in siCtrl or siERG-treated HUVEC ( n = 3 independent experiments). Graphical data presented in a and b are mean ± s.e.m., * P < 0.05, ** P < 0.01, Student’s t -test. c pcDNA, KLF2 or both KLF2 and ERG plasmids were co-transfected with TM WT promoter construct ( n = 4 independent experiments). Graphical data are mean ± s.e.m., *** P < 0.001, **** P < 0.0001, One-way ANOVA, compared to pcDNA-TM WT condition. d pcDNA or KLF2 plasmid were co-transfected with TM promoter constructs (TM WT, TM mutant 1 or TM mutant 2) ( n = 4 independent experiments). Graphical data are mean ± s.e.m., *** P < 0.001, **** P < 0.0001, One-way ANOVA. ## P < 0.01, compared to pcDNA-TM WT, Student’s t -test. e , f ChIP-qPCR using primers to region R1 and R2 on e ERG- or f KLF2-bound chromatin from siCtrl, siERG or siKLF2-treated HUVEC ( n = 3 independent experiments). Primers for a region within 5’UTR region of TM gene (TM neg con) were used as negative control. Data are shown as fold change over IgG. Graphical data are mean ± s.e.m., NS: Not Significant, * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t -test. g Representative image of proximity ligation assay (PLA) for nuclear ERG-KLF2 interaction was performed on confluent HUVEC using anti-ERG and anti-KLF2 antibodies; nuclei are identified by DAPI (blue). Scale bar, 20 μm. h Model: In homeostatic EC, ERG is required for p300 recruitment at the TM promoter region, leading to acetylation of H3K27 which opens the chromatin and allows KLF2 binding to the TM promoter. ERG binds to and cooperates with KLF2 to drive TM expression. In the absence of ERG, the chromatin is not accessible; KLF2 is not binding to the TM promoter and is not able to transactivate the TM promoter. Source data are provided as a Source Data file

Article Snippet: ERG (mouse) , Santa Cruz , sc-376293 , PLA (HUVEC, 1/200)/WB (HUVEC, 1/2000).

Techniques: Expressing, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Construct, Mutagenesis, ChIP-qPCR, Negative Control, Proximity Ligation Assay, Binding Assay

Journal: Nature Communications

Article Title: The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature

doi: 10.1038/s41467-019-12897-w

Figure Lengend Snippet: ERG drives thrombomodulin expression in vitro selectively in low shear stress conditions. a Representative image and quantification of ERG (green) and KLF2 (magenta) expression in HUVEC cultured under static conditions or exposed to low shear stress (LSS, 5 dynes per cm 2 ) or high shear stress (HSS, 20 dynes per cm 2 ) for 24 h by immunofluorescence; nuclei are identified by DAPI (blue). Scale bar 40 µm. Quantification represents the mean pixel intensity for ERG or KLF2 signal (arbitrary unit, A.U.) per cell ( n = 3–4 images per condition). b Representative image of proximity ligation assay (PLA) for nuclear ERG-KLF2 interaction was performed on confluent HUVEC cultured under static conditions or exposed to low shear stress (LSS, 5 dynes per cm 2 ) or high shear stress (HSS, 20 dynes per cm 2 ) for 24 h; nuclei are identified by DAPI (blue). Quantification represents the PLA signal (mean number of dots per cell) and is expressed as a percentage compared to static condition ( n = 3–4 images per condition). Scale bar, 40 μm. c qPCR analysis of ERG , KLF2 and TM mRNA expression in siCtrl or siERG-deficient HUVEC under static conditions or after 24 h under LSS or HSS ( n = 4). d qPCR analysis of ERG , KLF2 and TM mRNA expression in siCtrl or siERG-deficient HDBEC under static conditions or after 24 h under LSS or HSS ( n = 3 independent experiments). e Representative image and quantification of H3K27Ac expression (magenta) in siCtrl or siERG-deficient HUVEC cultured under static conditions or exposed to LSS or HSS for 24 h by immunofluorescence; nuclei are identified by DAPI (blue). Scale bar 40 µm. Quantification represents the mean pixel intensity for H3K27Ac signal (arbitrary unit, A.U.) per cell ( n = 6 images per condition). All graphical data are mean ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t -test for top panel and one-way Anova for bottom panel. Source data are provided as a Source Data file

Article Snippet: ERG (mouse) , Santa Cruz , sc-376293 , PLA (HUVEC, 1/200)/WB (HUVEC, 1/2000).

Techniques: Expressing, In Vitro, Shear, Cell Culture, Immunofluorescence, Proximity Ligation Assay