polyclonal antibody against egr1 sc110  (Santa Cruz Biotechnology)

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