polyclonal antibody against egr1  (Novus Biologicals)

Journal: PLoS ONE

Article Title: The Transcription Factor EGR1 Localizes to the Nucleolus and Is Linked to Suppression of Ribosomal Precursor Synthesis

doi: 10.1371/journal.pone.0096037

Figure Lengend Snippet: Imaging analysis. (A) Confocal images of EGR1 and fibrillarin (upper row) or EGR1 and B23 (lower row) in the nucleolus of HeLa cells. The images were obtained with a Leica SP2 and analyzed under HCX PL APO CS 63x. (B) Immunogold electron microscopy (EM) labeling of EGR1 in the fibrillar center of the nucleolus. Ultrathin sections of HeLa cells were embedded in Lowicryl K4M. (FC, Fibrillar center; DFC, Dense Fibrillar Component; GC, Granular Component. Arrow: labelling in the CF; arrowheads: labelling in the nucleoplasm).

Article Snippet: After a 4 h incubation with rabbit polyclonal antibody against N-terminal of EGR1 (Cell Signaling Technology, Danvers, MA, USA) diluted 1/2.5 in PBS containing 1/50 NGS and 0.2% BSA, the sections were washed with PBS containing 1% BSA, and incubated for 60 min with goat anti-rabbit IgG coupled to colloidal gold (10 nm in diameter) (Amersham Life Science) diluted 1/40 with PBS (pH 8.2) containing 0.2% BSA.

Techniques: Imaging, Electron Microscopy, Labeling

Journal: PLoS ONE

Article Title: The Transcription Factor EGR1 Localizes to the Nucleolus and Is Linked to Suppression of Ribosomal Precursor Synthesis

doi: 10.1371/journal.pone.0096037

Figure Lengend Snippet: Biochemical evidences. (A) Detection by immunoblotting of EGR1 in nuclei, nuclear and nucleolar extracts of HeLa grown at 0.2% or 10% FBS. The sumoylated form of EGR1 present within the nucleolar extracts is detected by an anti-sumo1 antibody (Sigma-Aldrich). The band signals were quantitated for comparison between extracts of cells grown at 0.2% and 10% FBS. Signals were normalized to the loading control. Beta-tubulin and fibrillarin are shown as loading control. (B) Immunofluorescence of EGR1 after treatment with actinomycin D (0.04 µg/ml) for 1h at 37°C. HeLa cells were treated and stained for EGR1 and fibrillarin by immunofluorescence. The images were taken by under a 40X objective with a LEICA DM4000B. The pictures at the right show the merging of the two fluorescing proteins. (C) Nuclei of HeLa cells were extracted and immunoblotted to quantitate the expression of EGR1 following Actinomycin D treatment. Both immunofluorescence and western blotting show that the endogenous levels of EGR1 are not significantly affected by the treatment. Representative results of at least three separate experiments are shown. Comparison tests were assessed by one way ANOVA, and significances are shown where applicable. Asterisk (*) represent p≤0.05 when compared to relative controls.

Article Snippet: After a 4 h incubation with rabbit polyclonal antibody against N-terminal of EGR1 (Cell Signaling Technology, Danvers, MA, USA) diluted 1/2.5 in PBS containing 1/50 NGS and 0.2% BSA, the sections were washed with PBS containing 1% BSA, and incubated for 60 min with goat anti-rabbit IgG coupled to colloidal gold (10 nm in diameter) (Amersham Life Science) diluted 1/40 with PBS (pH 8.2) containing 0.2% BSA.

Techniques: Western Blot, Comparison, Control, Immunofluorescence, Staining, Expressing

Journal: PLoS ONE

Article Title: The Transcription Factor EGR1 Localizes to the Nucleolus and Is Linked to Suppression of Ribosomal Precursor Synthesis

doi: 10.1371/journal.pone.0096037

Figure Lengend Snippet: Confocal images of HeLa cells transfected with (A) the full length EGR1 (1–543 AA), (B) the N-terminal (ΔC-EGR1) (1–314 AA), (C) the C-terminal EGR1 (ΔN-EGR1) (315–543 AA). Each construct was fused to the GFP. (D) Empty pEGFP vector. Full length EGR1, N-terminal EGR1, C-terminal EGR1 and stained with an antibody to fibrillarin.

Article Snippet: After a 4 h incubation with rabbit polyclonal antibody against N-terminal of EGR1 (Cell Signaling Technology, Danvers, MA, USA) diluted 1/2.5 in PBS containing 1/50 NGS and 0.2% BSA, the sections were washed with PBS containing 1% BSA, and incubated for 60 min with goat anti-rabbit IgG coupled to colloidal gold (10 nm in diameter) (Amersham Life Science) diluted 1/40 with PBS (pH 8.2) containing 0.2% BSA.

Techniques: Transfection, Construct, Plasmid Preparation, Staining

Journal: PLoS ONE

Article Title: The Transcription Factor EGR1 Localizes to the Nucleolus and Is Linked to Suppression of Ribosomal Precursor Synthesis

doi: 10.1371/journal.pone.0096037

Figure Lengend Snippet: (A) The synthesis of 47S rRNA is strongly upregulated following inhibition of EGR1. 47S synthesis in Hela cells grown in 0.2% FBS is significantly increased following endogenous EGR1 silencing with either 10 nM or 15 nM specific siRNA (middle graph). 47S synthesis is not affected in cells treated with a scrambled sequence compared to the untreated cells taken as control. The levels of expression of EGR1 and p300 following the siRNA treatment are shown in the left and right graphs, respectively. Both levels are significantly diminished after EGR1 silencing. (B) 47S synthesis in HeLa cells grown in 0.2% or 10% FBS is significantly depressed after transfection of full length EGR1. The levels of expression of EGR1 and p300 are shown in the left and right graphs, respectively. As expected, both levels are significantly upregulated after EGR1 transfection compared to control cells. Representative results of at least three separate experiments are shown. Comparison tests were performed by one way ANOVA, and significant results are highlighted with asterisks (* p≤0.05, ** p≤0.01 in comparison with relative controls).

Article Snippet: After a 4 h incubation with rabbit polyclonal antibody against N-terminal of EGR1 (Cell Signaling Technology, Danvers, MA, USA) diluted 1/2.5 in PBS containing 1/50 NGS and 0.2% BSA, the sections were washed with PBS containing 1% BSA, and incubated for 60 min with goat anti-rabbit IgG coupled to colloidal gold (10 nm in diameter) (Amersham Life Science) diluted 1/40 with PBS (pH 8.2) containing 0.2% BSA.

Techniques: Inhibition, Sequencing, Control, Expressing, Transfection, Comparison

Journal: PLoS ONE

Article Title: The Transcription Factor EGR1 Localizes to the Nucleolus and Is Linked to Suppression of Ribosomal Precursor Synthesis

doi: 10.1371/journal.pone.0096037

Figure Lengend Snippet: (A) The synthesis of 47S rRNA in NIH 3T3 (ARF−/−) cells grown in 0.2% FBS (right graph) is not affected when the expression of endogenous EGR1 is silenced with 10 nM of specific siRNA (left graph). (B) Viceversa, the synthesis of 47S rRNA (left graph) is greatly reduced when both EGR1 (middle graph) and p19ARF genes (right graph) are transfected and expressed in NIH 3T3 following transfection with plasmid expression vectors. Transfection with pEGFP is shown as control. Comparison tests were assessed by one way ANOVA, and significant differences are highlighted with asterisks (* p<0.05; ** p<0.01).

Article Snippet: After a 4 h incubation with rabbit polyclonal antibody against N-terminal of EGR1 (Cell Signaling Technology, Danvers, MA, USA) diluted 1/2.5 in PBS containing 1/50 NGS and 0.2% BSA, the sections were washed with PBS containing 1% BSA, and incubated for 60 min with goat anti-rabbit IgG coupled to colloidal gold (10 nm in diameter) (Amersham Life Science) diluted 1/40 with PBS (pH 8.2) containing 0.2% BSA.

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Comparison

Journal: PLoS ONE

Article Title: The Transcription Factor EGR1 Localizes to the Nucleolus and Is Linked to Suppression of Ribosomal Precursor Synthesis

doi: 10.1371/journal.pone.0096037

Figure Lengend Snippet: (A) Confocal analysis of Hela cells transfected with the C-terminal EGR1 (ΔN 1–314) shows the colocalization of EGR1 fragment with UBF. (B) Extracts (150 µg) of HeLa cells transfected with full length EGR1-GFP are immunoprecipitated with an antibody to UBF. (C) Chromatin precipitation assay. DNA fragments of ribosomal RNA promoter are immunoprecipitated with an antibody to EGR1 from extracts of HeLa cells transfected with full length EGR1. A six fold enrichment in ribosomal RNA promoter fragments was obtained from extracts of transfected cells compared to mock extracts. Representative results of at least three separate experiments are shown. Comparison tests were performed as above described (** p≤0.01).

Article Snippet: After a 4 h incubation with rabbit polyclonal antibody against N-terminal of EGR1 (Cell Signaling Technology, Danvers, MA, USA) diluted 1/2.5 in PBS containing 1/50 NGS and 0.2% BSA, the sections were washed with PBS containing 1% BSA, and incubated for 60 min with goat anti-rabbit IgG coupled to colloidal gold (10 nm in diameter) (Amersham Life Science) diluted 1/40 with PBS (pH 8.2) containing 0.2% BSA.

Techniques: Transfection, Immunoprecipitation, Comparison

Journal: Molecular and Cellular Biology

Article Title: Insulin Inhibits Lipolysis in Adipocytes via the Evolutionarily Conserved mTORC1-Egr1-ATGL-Mediated Pathway

doi: 10.1128/MCB.01584-12

Figure Lengend Snippet: Egr1 suppresses ATGL promoter activity. 293T cells were transfected with different lengths of mouse ATGL luciferase promoter constructs, together with eGFP. Cells were cotransfected with either Egr1 cDNA (A), Egr2 cDNA (B), or empty vector (EV). The luciferase activity in cell lysates was assayed as described in Materials and Methods and normalized based on the GFP fluorescence. Experiments were repeated three times, and data are presented for triplicate samples as means ± the SD. (C) Schematic representation of the proximal region of ATGL promoter with the consensus Egr1 binding site. Nucleotides that have been chosen for the site-directed mutagenesis are underlined. (D) Conserved nucleotides −44, −42, −41, −40, and −36 in the ATGL promoter (underlined in panel C) were substituted by T's as described in Materials and Methods. HEK293T cells were transfected with wild-type (wt) or mutant (mt) ATGL luciferase promoter constructs, together with eGFP. Cells were cotransfected with Egr1 or Egr2 or empty vector (EV) and analyzed as described for panels A and B. (E) Wild-type and TSC2−/− MEFs were treated with 100 nM rapamycin (Rap) for 48 h. Total cell lysates were analyzed by Western blotting for Egr1. Actin served as a loading control. *, P < 0.05; **, P < 0.01 (all panels).

Article Snippet: Polyclonal antibody against Egr1 (sc110) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activity Assay, Transfection, Luciferase, Construct, Plasmid Preparation, Fluorescence, Binding Assay, Mutagenesis, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Insulin Inhibits Lipolysis in Adipocytes via the Evolutionarily Conserved mTORC1-Egr1-ATGL-Mediated Pathway

doi: 10.1128/MCB.01584-12

Figure Lengend Snippet: Insulin-dependent Egr1 expression controls ATGL expression in 3T3-L1 adipocytes. (A) Differentiated 3T3-L1 adipocytes were treated with 100 nM insulin for the indicated periods of time. (Top panel) The levels of ATGL and Egr1 mRNA were determined in triplicate by quantitative PCR and normalized based on 36B4 mRNA. (Bottom panel) Total cell lysates were analyzed by Western blotting for Egr1 and ATGL. Actin served as a loading control. (B) 3T3-L1 adipocytes were treated with 100 nM insulin in the presence or absence of actinomycin D (AcD; 0.2 μg/ml) for 16 h. Total cell lysates were analyzed by Western blotting for ATGL. Perilipin served as a control for AcD action, and cellugyrin and actin served as loading controls. (C) 3T3-L1 adipocytes were infected with adenovirus expressing Egr1 (AdEgr1) and GFP (AdGFP) and cultured for 48 and 72 h. Total cell lysates were analyzed by Western blotting for Egr1 and ATGL. Actin served as a loading control. (D) 3T3-L1 adipocytes infected with adenovirus expressing Egr1 (AdEgr1) and GFP (AdGFP) and cultured for 48 h. Cells were then incubated in phenol red-free DMEM with 2% fatty acid-free BSA without (white bars) or with (black bars) 10 μM isoproterenol (Iso) for 2 h. Glycerol was measured in medium aliquots in triplicate and normalized by protein concentration in whole-cell lysates. Data are expressed as means ± the SD. (E) ChIP assays were performed in 3T3-L1 adipocytes treated with 100 nM insulin for 4 h. Genomic fragments were immunoprecipitated with antibody against Egr1 or rabbit IgG, amplified by PCR, separated in a 3% agarose gel, and visualized by ethidium bromide staining.

Article Snippet: Polyclonal antibody against Egr1 (sc110) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Infection, Cell Culture, Incubation, Protein Concentration, Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Staining

Journal: Molecular and Cellular Biology

Article Title: Insulin Inhibits Lipolysis in Adipocytes via the Evolutionarily Conserved mTORC1-Egr1-ATGL-Mediated Pathway

doi: 10.1128/MCB.01584-12

Figure Lengend Snippet: mTORC1 controls ATGL expression and lipolysis in 3T3-L1 adipocytes via Egr1. (A) 3T3-L1 adipocytes were treated with 100 nM insulin in the presence (+) or absence (−) of 100 nM rapamycin (Rapa) or PP242 (10 nM) for 16 h. Total cell lysates were analyzed by Western blotting. The dotted line indicates that irrelevant lanes have been spliced out. GAPDH served as loading control. (B) 3T3-L1 adipocytes were treated with 100 nM insulin in the presence (+) or absence (−) of 100 nM rapamycin (Rapa) or PP242 (10 nM) for 16 h. After that, cells were transferred to phenol red-free DMEM with 2% fatty acid free BSA without (white bars) or with (black bars) 10 μM isoproterenol (Iso) for 2 h. Glycerol was measured in medium aliquots in triplicate and normalized based on the protein concentration in whole-cell lysates. Data are expressed as means ± the SD. (C) 3T3-L1 adipocytes were incubated either in nutrient-free Krebs-Ringer-HEPES buffer (KRH) or in nutrient-enriched DMEM in the presence or absence of 100 nM insulin for 3 h. Levels of Egr1 mRNA were determined in triplicate by quantitative PCR and normalized by 36B4 mRNA. (D) 3T3-L1 adipocytes were incubated in nutrient-free Krebs-Ringer-HEPES buffer (KRH) for 3 h in the presence or absence of 5 mM leucine, 5 mM glucose, and 100 nM insulin as indicated. Total cell lysates were analyzed by Western blotting for phospho-4E-BP. Actin served as a loading control. The top panel shows the levels of Egr1 mRNA determined in triplicate by quantitative PCR and normalized based on the 36B4 mRNA. *, P < 0.05; **, P < 0.01 (all panels).

Article Snippet: Polyclonal antibody against Egr1 (sc110) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Western Blot, Protein Concentration, Incubation, Real-time Polymerase Chain Reaction

Journal: Molecular and Cellular Biology

Article Title: Insulin Inhibits Lipolysis in Adipocytes via the Evolutionarily Conserved mTORC1-Egr1-ATGL-Mediated Pathway

doi: 10.1128/MCB.01584-12

Figure Lengend Snippet: A high-fat diet activates mTORC1, increases the levels of Egr1, and decreases ATGL expression in epididymal white adipose tissue. (A) Male C57/BL6 mice were fed a low-fat or high-fat diet for 14 weeks; epididymal fat pads were dissected and frozen. Total lysates were analyzed by Western blotting. The panel shows two independent experiments with, respectively, 6 and 4 mice. (B) The levels of Egr1 mRNA were determined in triplicate by quantitative PCR and normalized based on the 36B4 mRNA levels. (C) Insulin and nutrients inhibit ATGL expression and lipolysis via the mTORC1-Egr1 regulatory axis.

Article Snippet: Polyclonal antibody against Egr1 (sc110) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction