Notes: To explore the interaction of liver receptor homolog (LRH)-1, a known suppressor of sterol regulatory element-binding protein (SREBP) transcriptional activity, human LRH-1 was reverse transcribed then amplified by PCR from total RNA from HepG2 cells. The amplification product was ligated into the pTargeT™ Mammalian Expression Vector to create pTarget-LRH1. For reporter experiments, a PCR fragment that encompassed the 1.3kb 5’-promoter region of the human small heterodimer partner (SHP) gene was cloned into the pGL3-Basic Vector (designated pSRB). The pGL3-Promoter Vector was used to construct pLRHREx3, which contains three LRH-1 response elements, and the insert was generated using synthetic oligonucleotides. HEK293 cells were cotransfected with 0.2µg of a promoter-firefly luciferase construct, 0.1µg of a SREBP expression plasmid, 10ng of phRL-TK Vector and 0.2 or 0.6µg of pTarget-LRH1. Alternatively, the cotransfected plasmids were 0.2µg of pSHP, 0.1µg of pTarget-LRH1, 10ng of phRL-TK Vector and 0.2 or 0.6µg of a SREBP expression plasmid. The pLRHREx3 construct (0.2µg) was cotransfected with 0.1µg of a LRH-1 expression plasmid, 0.2µg of pCMXPGC-1α (peroxisome proliferator activated receptor γ coactivator-1α), 10ng of phRL-TK Vector, and 0.1 or 0.3µg of a pSREBP expression vector in HEK 293 cells. Luciferase expression was assayed 48 hours post-transfection using the Dual-Luciferase^® Assay Reporter System. To express SREBPs and LRH-1 in vitro, inserts were ligated into the pTNT™ Vector, synthesized using the TNT^® Coupled Transcription/Translation System with radiolabeled methionine. Ten microliters of the ³⁵S-labelled protein was then used in a GST-pulldown assay. (3692)

Notes: The valosin-containing protein (VCP) is a target of Akt signaling. In this study, VCP was co-immunoprecipitated with Akt and shown to be phosphorylated at an Akt consensus site upon Akt activation by growth factors. Putative phosphorylation sites were identified on VCP, then site-directed mutagenesis was performed to investigate the importance of these sites in the VCP-Akt interaction. Wild-type and mutant VCP genes were cloned into the pCMVTNT™ vector and transfected into MCF-7 cells. The degree of phosphorylation of the Akt consensus site was then investigated in the presence or absence of mutant or wild-type VCP proteins. (3346)

Notes: This study investigated the role of c-Maf in transcriptional regulation of IL-10 in macrophages. c-Maf cDNA was cloned into the pTNT™ Vector, and recombinant human c-Maf synthesized from that plasmid using the TNT^® T7 Quick Coupled Transcription/Translation System. (3347)

Notes: This study showed that membrane type-1 matrix metalloproteinase (MT1-MMP) binds C1q, the recognition unit of complement C1, which activates the classical pathway of complement. The peptides involved in C1q binding were identified and found to be distinct from those involved in proteolysis. cDNA fragments encoding various peptide sequences were cloned into the pTNT™ Vector prior to expression in the TNT^® T7 Quick Coupled Transcription Translation System. Translation reaction products were used in a C1q binding assay. (3348)

Notes: This paper describes amplification of rat NHE1 cDNA using primers incorporating the Xho I and Xba I restriction endonuclease sites, Kozak consensus, ATG start site, and a stop codon. The resultant 1.1 kb NHE1 encoding-PCR product was digested with Xba I and Xho I and cloned into the pTNT™ Vector.  This construct was used as a template in a TNT^® T7 Quick Coupled Transcription/Translation reaction to generate substrates for an in vitro caspase-3 cleavage assay. Proteolytically cleaved fragments of NHE1 were run on a polyacrylamide gel and visualized by autoradiography. (3063)

Notes: Researchers cloned the Arabidopsis Cleavage Stimulation factors AtCst-77 and AtCst-64 into the pTNT™ Vector along with an N-terminus haemagglutinin (HA) epitope tag. These constructs were transcribed in vitro and the resultant mRNAs were translated and radiolabeled using Rabbit Reticulocyte Lysates. Interactions between AtCsf-77-HA and AtCsf-64-HA were studied by immunoprecipitation of the combined translation products using antibodies to AtCsf-64, AtCsf-77 or the HA tag. (2644)

Notes: Lymphoid Enhancer-Binding Factor-1 (LEF-1) cDNA was cloned into the pTNT™ Vector. One microgram of the resultant construct was used as a template for in vitro transcription/translation reactions using the TNT^® T3 Coupled Wheat Germ Extract System. Western blotting and gel shift assays verified the size and function of LEF-1 expressed from the reactions.  (2721)