Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: (A) Total cell lysates from MRC5 and other indicated NSCLC cell lines were immunoblotted with anti-EGR1. β-actin was used as loading control. (B) Representative of H1299 cells with or without overexpressed EGR1 immunofluorescence-stained with antibodies against EGR1 (red color). The nuclei were counterstained with DAPI (blue color). (C) The growth of H1299 cells infected with CD513B-1-EGR1 or -dnEGR1 was analyzed by CCK8 assay. Up panel: western blot for EGR1 expression levels in H1299 cells transfected with EGR1 or dnEGR1. (D) Histogram showing the percentages of apoptotic cells by flow cytometric staining with Annexin V-PE/7-AAD. (E) Western blot for apoptotic caspase-3 and -7 in H1299 cells with increasing quantities of EGR1 compared with control. Numbers presented the fold change compared with the vector. Relative values ± SD were obtained from three independent assays. The full-length blots of Fig. 1A, 1C and 1E were presented in .

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Immunofluorescence, Staining, Infection, CCK-8 Assay, Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: (A) The effect of EGR1 on cell migration was determined by wound healing assay. The spreading speed of EGR1-transfected cells along the wound edge was slower than that of vector- or dnEGR1-transfected cells over a period of 72 h. (C) Representative images showing the transfected cells that migrated through the cell. The number of migrated tumor cells was quantified and is shown in the right panel. Columns, mean of triplicate experiments; *P < 0.05.

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Migration, Wound Healing Assay, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: (A and B) Hierarchical clustering of 100 genes that exhibited significantly altered expression in EGR1-transfected H1299 cells as compared with vector-transfected H1299 cells. The color bar indicates the fold change (log 2 ). (C) IPA launch of bio-function enrichment profiles, plotted by relative statistical significance. P value, Fisher's exact test. (D) The expression of 15 upregulated genes (ARC, GDF15, CDKN1C, TGM2, HES1, EFNA1, NR4A1, TRIB1, IGFBP6, ITGB8, PDGFA, KRT18, TIMP1, and ID2) was analyzed by qPCR. Relative values ± SD were obtained from three independent assays.

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: Genes differentially expressed between EGR1-overexpressed H1299 cells and control cells

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Binding Assay, Dominant Negative Mutation, Expressing

Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: (A) Luciferase reporter assays of sequential deletions of the KRT18 promoter, cotransfected with increasing quantities of EGR1. The predicted positions of the EBS (black boxes) were indicated on the schematic. Nucleotides with red colors presented consensus EBS. Values are expressed as mean fold-activation to the promoter cotransfected with empty vector. (B) Luciferase reporter assays of point mutant of the KRT18 promoter. The consensus EBS in pGL3-KRT18 was mutated, creating pGL3-KRT18-mut. Relative reporter activity in response to EGR1 transfection was compared as in (A). (C) EGR1 and KRT18 protein levels after treatment with different si-EGR1 doses or NC (negative control). β-actin was used as loading control. Values are presented as fold change relative to NC. The full-length blot of Fig. 4C is presented in .

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Luciferase, Activation Assay, Plasmid Preparation, Mutagenesis, Activity Assay, Transfection, Negative Control

Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: (A) The growth of H1299 and A549 cells infected with CD513B-1-KRT18 was analyzed by CCK-8 assay. The results are expressed as mean ± SD of three independent experiments. (B) The effect of EGR1 on cell migration was determined by wound healing assay. (C) Representative images showing the transfected cells that migrated through the cell. The number of migrated tumor cells was quantified as in . (D) Western blot for apoptotic caspase-3 and -7 in H1299 cells with different KRT18 transfected dose as compared with control cells. β-actin was used as loading control. The full-length blot of Figure 5D is presented in . (E) Expression level of EGR1 and KRT18 in 2 primary NSCLC cases. (F) Correlation of the EGR1 (+) expression with age, lymph node metastasis and KRT18 in 36 NSCLC cases. (G) Correlation of the KRT18 (+) expression with lymph node metastasis in 36 NSCLC cases.

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Infection, CCK-8 Assay, Migration, Wound Healing Assay, Transfection, Western Blot, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: The Egr-1 gene is rapidly induced after TCR-mediated activation of the DPK double positive cell line. ( A ) Total RNA isolated from DPK cells cultured with immobilized anti-CD3ε mAb for the indicated times (shown in hours), was subjected to RT-PCR analysis using Egr-1 or CD4 primers. ( B ) Electrophoretic mobility shift assay using nuclear lysates prepared from DPK cells 8 h after activation by immobilized antiCD3ε mAb. Probes contained a single Egr-1 binding site ( left ) or overlapping Egr-1 and Sp1 sites ( right ). ( C ) DPK cells were cultured with DCEK-ICAM fibroblast antigen presenting cells and 1 μm pigeon cytochrome c peptide for the indicated times. Total RNA was isolated and subjected to a competitive RT-PCR assay (see Materials and Methods). Note the different scales for Egr-1 and CD4 mRNA expression.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Activation Assay, Isolation, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Electrophoretic Mobility Shift Assay, Binding Assay, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: DPK cell differentiation and Egr-2,3 mRNA induction is cyclosporin A sensitive, while Egr-1 mRNA induction is cyclosporin A resistant. ( A , B ) DPK cells were cultured with DCEK-ICAM fibroblast antigen presenting cells and 2 μM pigeon cytochrome c peptide in the presence or absence of 100 ng/ml cyclosporin A, or appropriate dilution of solvent (DMSO) as indicated. Cells were harvested and stained for CD69 after 1 or 3 d in culture ( A ) or stained for CD4 and CD8 after 3 d in culture ( B ). ( C ) RT-PCR analysis of total RNA derived from DPK cells activated for 6 h with immobilized anti-CD3ε mAb in the presence or absence of 300 ng/ml cyclosporin A, using Egr-1, Egr-2 (Krox-20), CD4, CD69 or Egr-3 primers. (*) Also shown for the indicated samples is the relative level of Egr-1 cDNA normalized to expression of CD4 cDNA as determined by competitive RT-PCR assay. The identity of the lower major band in Egr-3 RT-PCR was verified by sequencing.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Cell Differentiation, Cell Culture, Staining, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Expressing, Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Expression of Egr gene family is dependent upon ras signaling pathways. ( A ) Competitive RT-PCR was used to compare expression of Egr-1 and CD4 genes in DPK or 17N4 cells activated for 6 h with immobilized anti-CD3ε mAb. ( B ) RT-PCR analysis of total RNA derived from DPK or 17N4 cells activated for 6 h with immobilized anti-CD3ε mAb. Independent PCR reactions using Egr-2 (Krox-20), CD4, or Egr-3 primers were performed.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Egr-1 mRNA and DNA binding activity in the thymus is MHC dependent. ( A ) Thymocytes derived from wild-type or MHCdeficient mice were two color-stained for CD4 and CD8. ( B ) Competitive RT-PCR was used to determine the level of expression of CD4 and Egr-1 genes in thymocytes derived from wild-type or MHC-deficient mice. ( C ) Electrophoretic mobility shift assay using nuclear lysates prepared from freshly isolated thymocytes derived from wild-type or MHCdeficient mice using a probe containing an Egr-1 binding site. Nuclear extracts derived from 5 × 10 5 cells containing equivalent amounts of protein were used in binding reactions. In some instances as indicated, binding reactions contained anti-Egr-1 antibody or normal rabbit serum (NRS). For comparison, a binding reaction containing recombinant Egr-1 is shown.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Binding Assay, Activity Assay, Derivative Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Electrophoretic Mobility Shift Assay, Isolation, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Expression of Egr-1 mRNA in double positive thymocytes. ( A ) Total thymocytes and CD4 + 8 + thymocytes (isolated by cell sorting, 95% DP) derived from the same animal, were assayed for expression of Egr and CD4 mRNA by competitive RT-PCR. Shown is the relative level of Egr-1 cDNA in the sample, normalized to the level of CD4 cDNA. ( B ) Total thymocytes derived from an MHC-deficient mouse were cultured with hamster immunoglobulin-coated or anti-CD3ε mAbcoated beads for 90 min before determination of Egr-1 and CD4 gene expression as in Fig. C .

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Expressing, Isolation, FACS, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Expression of Egr-1 protein in the thymus. Thin sections of normal thymus ( A–C ) or MHC knockout thymus ( D ) were fixed in formaldehyde and stained with a specific rabbit anti-Egr-1 peptide antiserum ( A , C , D ) or the same antibody preincubated with specific peptide ( B ). Regions of cortex ( C ) and medulla ( M ) are indicated. Sections were counterstained with hematoxylin and photographed at ×20 ( A , B ) or ×40 ( C , D ). C shows a magnification of the same section photographed in A .

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Expressing, Knock-Out, Staining

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Expression of Egr-1 protein in thymocyte subsets. Thymocytes from a young adult mouse were 4-color stained for expression of CD4, CD8, Egr-1, and CD69 or CD3, and analyzed by FACS ® as described in Materials and Methods. Indicated in the dot plots are the percentage of thymocytes within each quadrant, or in parenthesis ( upper right dot plot ), the percentage of Egr-1 + thymocytes within each thymocyte subset. Where indicated, staining is shown for gated populations of thymocytes (either Egr-1 + thymocytes as shown in histogram, or CD4 + 8 + thymocytes). Similar results were obtained from three other individual animals.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Expressing, Staining

Journal: Oncotarget

Article Title: Prostaglandins induce early growth response 1 transcription factor mediated microsomal prostaglandin E 2 synthase up-regulation for colorectal cancer progression

doi:

Figure Lengend Snippet: A. mPGES1 expression is reduced by aspirin (ASP, 1 μM) or etoricoxib (ETOR, 1 μM) treatment for 24 h, in both HT29-LucD6 EV and COX2 cells as quantified by qRT-PCR. B. PTGES mRNA is increased by PGF 2α (1 μM) in EV cells while it is reduced by AL8810 (10 μM) treatment (24 h) in HT29-LucD6-COX2. C, D. Relative luciferase activity of cells transfected with the “full length” (PTGES-A, -631 to -1) and “proximal” (PTGES-B, -177 to-1) constructs of the human PTGES promoter transfected in HT29-EV and –COX2 cells. E. mRNA levels of EGR1 were quantified by RT-PCR in samples from EV and COX2-overexpressing HT29-LucD6 and SW620 cells. F. Relative luciferase activity of an EGR1 reporter construct EV and COX2 in HT29 and SW480ADH cells. EGR1 mRNA G. and protein H. induction after 24 h treatment of the indicated cell lines with 1 μM of the PGs shown. * p < 0,005

Article Snippet: Membranes were incubated with specific antibodies against COX2, mPGES1 (Cayman Chemicals Europe) or anti- EGR1 (sc-189) or anti-HSP90 (sc-7947) antibody (Santa Cruz Biotechnology).

Techniques: Expressing, Quantitative RT-PCR, Luciferase, Activity Assay, Transfection, Construct, Reverse Transcription Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Prostaglandins induce early growth response 1 transcription factor mediated microsomal prostaglandin E 2 synthase up-regulation for colorectal cancer progression

doi:

Figure Lengend Snippet: A. Relative levels of PTGS2 , EGR1 and PTGES mRNA, estimated by RT-PCR analysis of HT29-EV and –COX2 cells transfected with 3 different shRNA expressing plasmids, specific for EGR1 or one shRNA without any known mammalian target (scr). B. Immunofluorescence for mPGES1 and EGR1 of HT29-EV and –COX2 cells transfected as mentioned above. C. Fluorescence quantification averages of the images of three independed experiments, as in (B) * p < 0,001. Bar: 32 μm.

Article Snippet: Membranes were incubated with specific antibodies against COX2, mPGES1 (Cayman Chemicals Europe) or anti- EGR1 (sc-189) or anti-HSP90 (sc-7947) antibody (Santa Cruz Biotechnology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, shRNA, Expressing, Immunofluorescence, Fluorescence

Journal: Oncotarget

Article Title: Prostaglandins induce early growth response 1 transcription factor mediated microsomal prostaglandin E 2 synthase up-regulation for colorectal cancer progression

doi:

Figure Lengend Snippet: Pearson's correlation coefficient calculated from the gene expression data for the genes shown in the two colorectal tumor collections (Gaedcke colorectal and Skrzypczak Colorectal 2; Oncomine)

Article Snippet: Membranes were incubated with specific antibodies against COX2, mPGES1 (Cayman Chemicals Europe) or anti- EGR1 (sc-189) or anti-HSP90 (sc-7947) antibody (Santa Cruz Biotechnology).

Techniques: Gene Expression

Journal: Molecular carcinogenesis

Article Title: CtBP2 Regulates Wnt Signal Through EGR1 to Influence the Proliferation and Apoptosis of DLBCL Cells.

doi: 10.1002/mc.23901

Figure Lengend Snippet: FIGURE 5 | Interfering with EGR1 inhibited the proliferation and cycle acceleration of DLBCL cells and promotes apoptosis. The EGR1 gene in cells was silenced through siRNA transfection. (A) The interference level of EGR1 was measured by western blot analysis. (B) Cell proliferation was detected by CCK8 assay. (C) EdU staining was performed to evaluate the level of cell proliferation; Flow cytometry was conducted to analyze the cell cycle (D) and the level of apoptosis (E). (F) The expressions of CtBP2 and apoptosis‐related proteins were detected by western blot. ***p < 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The antibodies utilized in this study included CtBP2 (10346‐1‐AP; Proteintech), EGR‐1 (22008‐1‐AP; Proteintech), Bcl‐2 (12789‐1‐AP; Proteintech), Bax (ab32503; Abcam), Caspase 3 (9961, CST), cleaved‐Caspase 3 (9962, CST), DKK1 (ab307367; Abcam), β‐catenin (ab32572; Abcam) and c‐Myc (ab185656; Abcam).

Techniques: Transfection, Western Blot, CCK-8 Assay, Staining, Flow Cytometry

Journal: Molecular carcinogenesis

Article Title: CtBP2 Regulates Wnt Signal Through EGR1 to Influence the Proliferation and Apoptosis of DLBCL Cells.

doi: 10.1002/mc.23901

Figure Lengend Snippet: FIGURE 6 | Interfere with CtBP2 inhibited the proliferation and cycle acceleration of DLBCL cells and promoted apoptosis through EGR1. The cells were transfected with an Ov‐EGR1 overexpression plasmid. (A) The overexpression level of EGR1 was assessed using Western blot analysis. (B) Cell proliferation was evaluated by CCK‐8 assay. (C) Cell proliferation was measured through EdU staining; Flow cytometry was performed to analyze the cell cycle (D) and the level of apoptosis (E). (F) The expression of apoptosis‐related proteins was detected by western blot assay. ***p < 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The antibodies utilized in this study included CtBP2 (10346‐1‐AP; Proteintech), EGR‐1 (22008‐1‐AP; Proteintech), Bcl‐2 (12789‐1‐AP; Proteintech), Bax (ab32503; Abcam), Caspase 3 (9961, CST), cleaved‐Caspase 3 (9962, CST), DKK1 (ab307367; Abcam), β‐catenin (ab32572; Abcam) and c‐Myc (ab185656; Abcam).

Techniques: Transfection, Over Expression, Plasmid Preparation, Western Blot, CCK-8 Assay, Staining, Flow Cytometry, Expressing

Journal: Molecular carcinogenesis

Article Title: CtBP2 Regulates Wnt Signal Through EGR1 to Influence the Proliferation and Apoptosis of DLBCL Cells.

doi: 10.1002/mc.23901

Figure Lengend Snippet: FIGURE 7 | EGR1 may be involved in the regulation of the Wnt/β‐catenin signaling pathway by CtBP2. The expressions of Wnt/β‐catenin signaling‐related proteins DKK1, β‐catenin and c‐MyC were detected by western blot. ***p < 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The antibodies utilized in this study included CtBP2 (10346‐1‐AP; Proteintech), EGR‐1 (22008‐1‐AP; Proteintech), Bcl‐2 (12789‐1‐AP; Proteintech), Bax (ab32503; Abcam), Caspase 3 (9961, CST), cleaved‐Caspase 3 (9962, CST), DKK1 (ab307367; Abcam), β‐catenin (ab32572; Abcam) and c‐Myc (ab185656; Abcam).

Techniques: Western Blot

Journal: The Journal of biological chemistry

Article Title: Sp1 and egr-1 have opposing effects on the regulation of the rat Pgp2/mdr1b gene.

doi: 10.1074/jbc.274.5.3199

Figure Lengend Snippet: FIG. 2. Sp1 activates and is essen- tial for full pgp2/mdr1b promoter ac- tivity. A, radiolabeled pgp2GC or mtpgp- 2GC oligonucleotide was incubated with (1) and without (2) affinity-purified Sp1 (one footprinting unit) and analyzed by EMSA. The last lane with mtpgp2GC con- tains 5 footprinting units of Sp1. B, the activity of 2250WT promoter was com- pared with the 2250MT pgp2/mdr1b mu- tant promoter that contained the identi- cal mutations as were found in the mtpgp2GC oligonucleotide. 10 mg of each of these plasmids were transiently trans- fected into H35 cells and luciferase activ- ity measured. Results are expressed as activity relative to the 2250WT promoter (100%). C, Drosophila Schneider cells were transiently transfected with 1 mg of the 2250WT or 2250MT plasmid along with indicated amounts of pPacSp1 (wild- type Sp1 expression plasmid) or N539 (a COOH-terminal Sp1 deletion mutant) and luciferase activity measured. Values in B and C are the average of three sepa- rate transfection experiments each per- formed in duplicate to quadruplicate. The error bars represent the standard error of the mean. Results are expressed as fold- activation relative to 2250WT (1.0) or 2250MT (1.0) co-transfected with empty vector.

Article Snippet: Polyclonal antibodies to Egr-1 and Sp1 were purchased from Santa Cruz Biotech, and the mdr1 (Ab-1) and actin antibodies were purchased from Calbiochem (La Jolla, CA).

Techniques: Incubation, Affinity Purification, Footprinting, Activity Assay, Luciferase, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Activation Assay

Journal: The Journal of biological chemistry

Article Title: Sp1 and egr-1 have opposing effects on the regulation of the rat Pgp2/mdr1b gene.

doi: 10.1074/jbc.274.5.3199

Figure Lengend Snippet: FIG. 3. Pgp and Sp1 levels and Sp1 DNA binding in H35 clones stably ex- pressing Sp1 (Sp1–9, 10, 18, or 20) or Neo vector (pc2). A, analysis of Sp1: 50 mg of nuclear extract protein from the in- dicated cell lines was analyzed by immu- noblot with anti-Sp1 IgG. B, a radiola- beled consensus Sp1 oligonucleotide was incubated with nuclear extracts prepared from H35 clones stably expressing Sp1 or pcDNA vector and analyzed by EMSA. C, analysis of Pgp: 35 mg of total cell lysate protein from H35 clones was analyzed by immunoblot.

Article Snippet: Polyclonal antibodies to Egr-1 and Sp1 were purchased from Santa Cruz Biotech, and the mdr1 (Ab-1) and actin antibodies were purchased from Calbiochem (La Jolla, CA).

Techniques: Binding Assay, Clone Assay, Stable Transfection, Plasmid Preparation, Incubation, Expressing, Western Blot

Journal: The Journal of biological chemistry

Article Title: Sp1 and egr-1 have opposing effects on the regulation of the rat Pgp2/mdr1b gene.

doi: 10.1074/jbc.274.5.3199

Figure Lengend Snippet: FIG. 4. Pgp2/mdr1b binds authentic Egr-1 and Egr-1 in H35 Cells. A, 32P end-labeled pgp2GC oligonucleotide was incubated with H35 nuclear extract in the presence or absence (NC, no competitor) of excess unlabeled pgp2GC, Sp1, Egr, or Oct-1 oligonucleotide (see “Ex- perimental Procedures” for amounts) and analyzed by EMSA. B, 32P- labeled pgp2GC or Egr consensus oligonucleotides were incubated with in vitro translated Egr1 in the presence or absence (NC, no competitor) of various competitor oligonucleotides. The molar excess of competitors was as follows: Oct-1 (200-fold), pgp2GC (100-fold), EgrWT (100-fold), and EgrMT (200-fold).

Article Snippet: Polyclonal antibodies to Egr-1 and Sp1 were purchased from Santa Cruz Biotech, and the mdr1 (Ab-1) and actin antibodies were purchased from Calbiochem (La Jolla, CA).

Techniques: Labeling, Incubation, In Vitro

Journal: The Journal of biological chemistry

Article Title: Sp1 and egr-1 have opposing effects on the regulation of the rat Pgp2/mdr1b gene.

doi: 10.1074/jbc.274.5.3199

Figure Lengend Snippet: FIG. 6. Competitive displacement of Sp1 binding to the 236 GC-rich region in the Pgp2/mdr1b promoter by Egr-1. EMSA was performed on 32P end-labeled pgp2GC oligonucleotide (left panel) incu- bated in the presence of a constant amount of Sp1 (1 footprinting unit/incubation) and increasing amounts of bacterially expressed Egr- 1-GST fusion protein, or (right panel) incubated with a fixed amount of recombinant Egr-1-GST (2 ml/reaction) and increasing amounts of re- combinant Sp1 (1–10 footprinting units).

Article Snippet: Polyclonal antibodies to Egr-1 and Sp1 were purchased from Santa Cruz Biotech, and the mdr1 (Ab-1) and actin antibodies were purchased from Calbiochem (La Jolla, CA).

Techniques: Binding Assay, Labeling, Footprinting, Incubation, Recombinant

Journal: The Journal of biological chemistry

Article Title: Sp1 and egr-1 have opposing effects on the regulation of the rat Pgp2/mdr1b gene.

doi: 10.1074/jbc.274.5.3199

Figure Lengend Snippet: FIG. 8. Enforced expression of Egr-1 in H35 hepatoma cells affects mdr1 expression. H35 cells were transfected with either the Egr-1 expression vector or pcDNA3, placed under G418 selection and Egr-Neo and Neo clones isolated, expanded and characterized by West- ern analysis for Egr-1 and Sp1 expression. The loading control was a nonspecific band that appears equivalent in all samples.

Article Snippet: Polyclonal antibodies to Egr-1 and Sp1 were purchased from Santa Cruz Biotech, and the mdr1 (Ab-1) and actin antibodies were purchased from Calbiochem (La Jolla, CA).

Techniques: Expressing, Transfection, Plasmid Preparation, Selection, Clone Assay, Isolation, Control