rabbit polyclonal antibodies against egr1  (Santa Cruz Biotechnology)

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