Netune hepatic metabolic control.overexpression of HA-tagged lipin 1 proteins were performed

Netune hepatic metabolic control.overexpression of HA-tagged lipin 1 proteins were performed with mouse monoclonal anti-HA antibody (Covance). Mouse anti actin antibody was purchased from Sigma Chemical Co.Chromatin Immunoprecipitation (ChIP) AssaysIn experiments where ChIP was the endpoint, HepG2 cells were cultured in 10 cm dishes and infected with Ad-GFP, AdHNF4a, and/or Ad-lipin 1b. Approximately 48 h after infection, proteins were cross-linked to chromatin by adding formaldehyde to a final concentration of 1 and incubating for 15 minutes at room temperature. Chromatin purification and ChIP assays were performed by using a commercially available ChIP assay kit (Upstate Biotechnology) according to the manufacturer’s instructions. PCR primers were designed to flank HNF4a response elements and the sequence of these primers is available upon request. For quantification, SYBR GREEN PCR was performed using DNA obtained from ChIP.Methods Mouse StudiesAll animal experiments were approved by the Animal Studies Committee of Washington University School of Medicine. C57BL/6 mice were generated from a colony established in the Washington University mouse facility. Mice constitutively deficient in lipin 1 (fld mice), were compared to wild-type (+/+) littermate control mice (Balb/cByJ strain).DNA ConstructsLuciferase reporter driven by 2045 bp of the 59 flanking ML-240 region of the Lpin1 gene (22045.Lpin1.Luc) was the gift of Karen Reue and the +2293.Lpin1.Luc, which contains 393 nucleotides of the 59 flanking sequence, the first Lpin1 exon (51 nucleotides), and approximately 2,242 bp of the first intron of the mouse Lpin1 gene into a promoterless luciferase reporter vector (+2293-Lpin1.luc), has been recently described [13]. Ppara.Luc and Acadm.TKLuc were the gift of Bart Staels and Daniel Kelly, respectively, and have been reported and characterized previously [14,15]. The homologous promoter-firefly luciferase reporter construct driven by the entire intergenic region between the Apoc3 and Apoa4 genes (Apoc3/Apoa4.Luc) has been described [16,17] and was the generous gift of V. Giguerre. Apoc3/Apoa4 promoter luciferase reporter constructs containing mutations in HNF4a response elements were derived from the parent Apoc3/Apoa4.Luc construct and were provided by B. Spiegelman [16]. The Apoc3 enhancer.3X.Luc heterologous reporter construct was driven by the thymidine (-)-Indolactam V chemical information kinase minimal promoter and three copies of the human Apoc3 enhancer (59-GATCTCCCAGGGCGCTGGGCAAAGGTCACCTGCTGACCAGTGGAGATGAG-39; nuclear receptor response element is underlined). The UAS-TKLuc has also been previously described [10]. The SV40-driven renilla luciferase DNA construct was obtained from Promega. The 924 amino acid form of mouse lipin 1 (lipin 1b) fused to an N-terminal triple HA tag was overexpressed using a pCDNA3.1 vector [10]. Expression constructs driving expression of lipin 1b protein with a site-directed mutation changing isoleucine 726 and leucine 727 to phenylalanine (LXXFF) were derived 1379592 from the lipin 1b construct [4,10]. The HNF4a (pMT-HNF4a), PGC-1a, PGC1a-mL2 (LXXLL to LXXFF), and PGC-1b expression constructs have also been described [18,19]. The Gal4-HNF4a expression construct has also been described [20]. Adenoviral constructs to drive expression of HA-tagged lipin1b and/or GFP [4,10], Ad-HNF4a [16], and mutant lipin 1b (LXXFF) [2] have been previously described. Adenoviral transductions were carried out by incubating hepatocytes with adenoviruses in a 5 FBS/DMEM.Netune hepatic metabolic control.overexpression of HA-tagged lipin 1 proteins were performed with mouse monoclonal anti-HA antibody (Covance). Mouse anti actin antibody was purchased from Sigma Chemical Co.Chromatin Immunoprecipitation (ChIP) AssaysIn experiments where ChIP was the endpoint, HepG2 cells were cultured in 10 cm dishes and infected with Ad-GFP, AdHNF4a, and/or Ad-lipin 1b. Approximately 48 h after infection, proteins were cross-linked to chromatin by adding formaldehyde to a final concentration of 1 and incubating for 15 minutes at room temperature. Chromatin purification and ChIP assays were performed by using a commercially available ChIP assay kit (Upstate Biotechnology) according to the manufacturer’s instructions. PCR primers were designed to flank HNF4a response elements and the sequence of these primers is available upon request. For quantification, SYBR GREEN PCR was performed using DNA obtained from ChIP.Methods Mouse StudiesAll animal experiments were approved by the Animal Studies Committee of Washington University School of Medicine. C57BL/6 mice were generated from a colony established in the Washington University mouse facility. Mice constitutively deficient in lipin 1 (fld mice), were compared to wild-type (+/+) littermate control mice (Balb/cByJ strain).DNA ConstructsLuciferase reporter driven by 2045 bp of the 59 flanking region of the Lpin1 gene (22045.Lpin1.Luc) was the gift of Karen Reue and the +2293.Lpin1.Luc, which contains 393 nucleotides of the 59 flanking sequence, the first Lpin1 exon (51 nucleotides), and approximately 2,242 bp of the first intron of the mouse Lpin1 gene into a promoterless luciferase reporter vector (+2293-Lpin1.luc), has been recently described [13]. Ppara.Luc and Acadm.TKLuc were the gift of Bart Staels and Daniel Kelly, respectively, and have been reported and characterized previously [14,15]. The homologous promoter-firefly luciferase reporter construct driven by the entire intergenic region between the Apoc3 and Apoa4 genes (Apoc3/Apoa4.Luc) has been described [16,17] and was the generous gift of V. Giguerre. Apoc3/Apoa4 promoter luciferase reporter constructs containing mutations in HNF4a response elements were derived from the parent Apoc3/Apoa4.Luc construct and were provided by B. Spiegelman [16]. The Apoc3 enhancer.3X.Luc heterologous reporter construct was driven by the thymidine kinase minimal promoter and three copies of the human Apoc3 enhancer (59-GATCTCCCAGGGCGCTGGGCAAAGGTCACCTGCTGACCAGTGGAGATGAG-39; nuclear receptor response element is underlined). The UAS-TKLuc has also been previously described [10]. The SV40-driven renilla luciferase DNA construct was obtained from Promega. The 924 amino acid form of mouse lipin 1 (lipin 1b) fused to an N-terminal triple HA tag was overexpressed using a pCDNA3.1 vector [10]. Expression constructs driving expression of lipin 1b protein with a site-directed mutation changing isoleucine 726 and leucine 727 to phenylalanine (LXXFF) were derived 1379592 from the lipin 1b construct [4,10]. The HNF4a (pMT-HNF4a), PGC-1a, PGC1a-mL2 (LXXLL to LXXFF), and PGC-1b expression constructs have also been described [18,19]. The Gal4-HNF4a expression construct has also been described [20]. Adenoviral constructs to drive expression of HA-tagged lipin1b and/or GFP [4,10], Ad-HNF4a [16], and mutant lipin 1b (LXXFF) [2] have been previously described. Adenoviral transductions were carried out by incubating hepatocytes with adenoviruses in a 5 FBS/DMEM.