Gata4 c 20

Mammalian expression vector pIRES(+)-GATA4 was used to perform DpnI-mediated site-directed mutagenesis to generate GATA4 N273K (c.819C>A) mutation. Two independent clones were created, sequenced, and assayed in transient transfection assays in quadruplicates on three independent experiments, as described (15 (link)). Lipofectamine 2000 was used to transfect HeLa cells with 0.15μg of pGL3-WNT2 promoter-Luciferase construct (16 (link)), 2ng pRL Renilla Luciferase reporter vector, in conjunction with different amounts of empty vector (pIRES-hrGFP), pIRES-GATA4 or pIRES-GATA4-N273K to reach 0.5μg total DNA using. Firefly and Renilla luciferase activity was assayed using Dual-Luciferase Reporter Assay System (Promega). Binding of nuclear lysates from cells expressing GATA4 and GATA4-N273K protein to P³²-labeled oligonucleotides including binding sites for GATA4 was performed as described (15 (link)). Sequences of oligonucleotides used in this assay include a predicted GATA binding site in the pancreatic (P2) HNF4A proximal promoter. Specificity of retardation complex was assessed by preincubating nuclear extracts with 100-fold excess wild type or mutant unlabeled oligonucleotides, or GATA4 antiserum (GATA4 C-20, Santa Cruz, sc-1237). Immunoblot analysis was used to verify that the expression efficiencies for vectors encoding wild type and N273K GATA4 were similar.