Notes: Wildtype and mutant androgen receptors (AR) were produced using the TNT^® Coupled Reticulocyte Lysate System (in some reactions in the presence of [³⁵S]methionine). In gel-retardation studies, the presence of agonists increases the mobility of AR-ARE complexes on nondenaturing gels. In partial proteolysis studies, agonists protect a 30kDa fragment from proteolysis. (0953)

Notes: [³⁵S]methionine-labeled NUC-1 proteins were expressed in the TNT^® SP6 Coupled Reticulocyte Lysate System. Gel shift mobility assays were performed using the in vitro synthesized NUC-1 proteins and DNA from the pho-4+ gene. Wild type NUC-1 specifically interacts with the sequence tested. Following immunoprecipitation of NUC-1 proteins, chemical cross-linking was performed using the same DNA as before and indicates that NUC-1 forms homodimers. (1859)

Notes: The reactivity of antibodies from patients suffering from systemic autoimmune diseases was examined. Deletion mutants of recombinant L7 were expressed in the TNT^® Coupled Reticulocyte Lysate System. Sera were collected from individuals suffering from systemic autoimmune diseases and were applied to immunoblots carrying total cell lysates. Immunoblots revealed that specific epitopes were often targets of antibodies in multiple patients, although the humoral autoimmune response and reactivity pattern of patient sera were heterogeneous. (1776)

Notes: [³⁵S]methionine-labeled protein was produced from the largest MSG cDNA clone and the viral gene 9 linked by a Factor Xa site using the TNT^® Coupled Reticulocyte Lysate System. The product was digested with Factor Xa, and it was immunoprecipitated with MSG antibodies. There may be multiple MSG mRNAs that were transcribed, but it is not known if they are all translated. (0770)

Notes: HCR (heme-controlled repressor) was produced using the TNT^® Coupled Reticulocyte Lysate System and found to migrate on SDS-PAGE with an apparent mass of 85kDa instead of the predicted size of ~70kDa. The slower migration may be due to phosphorylation. (0677)

Notes: [³⁵S]-labeled rabbit apoB mRNA editing protein (REPR) was generated using the TNT^® Coupled Reticulocyte Lysate System. The protein was tested for mRNA editing of the apoB RNA substrate. The protein only edited the apoB mRNA when chicken enterocyte extract was added. (0134)

Notes: Mouse germ cell nuclear factor (mGCNF) was cloned from cDNA libraries. The hypothesized role for this protein is regulating gene expression during meiosis. mGCNF was synthesized using the TNT® T3 Coupled Reticulocyte Lysate System. Gel mobility analyses were performed with in vitro synthesized mGCNF in the absence and presence of labeled and cold DRO (direct repeats with 0 nucleotide spacing) and SF-1, HS and Oct-1 unlabeled nucleotides. GCNF can bind to an SF-1 half-site but not to an NBRE half-site. (1828)

Notes: Full-length and fragments of [35S]methionine-labeled U1 snRNP-A protein were produced using the TNT® Coupled Reticulocyte Lysate System. The proteins were used in RNA-binding assays to the U1 RNA, SV40 RNA, pGEM® RNA (negative control) and triple mutant of SV40 sequences. The results show that the U1 snRNP-A is able to coprecipitate the U1 RNA and SV40 RNA, but a ten-fold reduction in precipitation of the triple mutant is seen. Both U1 fragments could bind the mutant but at greatly reduced levels. One U1 fragment precipitated both the U1RNA and the SV40 RNA; the other U1 fragment only precipitated the SV40 RNA. (1837)

Notes: Specific protein-protein interactions were mapped in vitro using a variety of techniques, including co-immunoprecipitation. dTAFII150DeltaN, dTAFII150DeltaC, pTAF150DeltaN and pTAF150DeltaC were expressed in the TNT^® Coupled Reticulocyte Lysate System in the presence of ³⁵S label. Protein A-Sepharose^® beads were bound to antibodies to dTAFII250 or to the HA epitope and incubated with HA-dTBP, HA-dTAFII110 and dTAFII250DeltaN fusion proteins expressed in baculovirus. The ³⁵S-labeled proteins were incubated with the antibody-baculovirus-expressed protein complexes and then analyzed by SDS-PAGE. dTAFII150 was shown to bind efficiently to immobilized HA-tagged dTBP and dTAFII250DeltaN, but did not interact selectively with dTAFII110 in vitro. (1775)

Notes: Wild type and mutant ecNOS (endothelial nitric oxide synthase) were synthesized in the TNT^® Coupled Reticulocyte Lysate System in the presence of [³H]myristic acid. [³H] was incorporated into the wild type protein verifying that the in vitro synthesized protein undergoes myristolation. ecNOS was also synthesized in the presence of [³⁵S]methionine and used in membrane association assays. ecNOS associates with the biological and denatured membranes. (1862)

Notes: All proteins used in the study were synthesized using TNT® SP6 Coupled Reticulocyte Lysate System and were used for EMSAs. Extradenticle, a DNA binding partner of certain homeotic proteins, binds cooperatively with Ultrabithorax, Abdominal-A or Engrailed proteins to the oligonucleotide probe used in these studies. The Engrailed-Extradenticle binding site is distinct from that bound by Extradenticle-Ultrabithorax or Extradenticle-Abdominal-A. Extradenticle does not interact with Abdominal-B. (1834)

Notes: [35S]labeled Groucho protein was synthesized in vitro using the TNT® Coupled Reticulocyte Lysate System. The protein was incubated with GST, GST-hairy, GST-E(spl)-m7, GST-E(spl)-m8 and GST-T4 on glutathione beads. Gro interacts with all of the GST fusion proteins except GST-T4 (and the GST control). (1809)

Notes: [³⁵S]Methionine-labeled RalGDS (guanine nucleotide dissociation stimulator) was transcribed and translated in the TNT^® T7 Coupled Reticulocyte Lysate System. The lysate was incubated with GTPase samples and used in immunoprecipitation assays with anti-RalGDS antisera. GST-R-ras, GST-H-ras and GST-Rap (Ras-like GTPases) bound with full length RalGDS in a GTP-dependent manner. In addition, Raf and RalGDS were shown to compete for binding to Ras-like GTPases. (1416)

Notes: [³⁵S]-methionine-labeled pRB60, pRB50 and pRB105 proteins were synthesized using TNT® Coupled Reticulocyte Lysate System. Experiments were performed with these proteins and GST fusion proteins to determine protein-binding sites. At low levels of GST-fusion protein, the pRB60 protein bound immobilized GST-E7(3-98) protein but did not bind the GST-E7-CR3 fusion or GST. The other truncated pRB translation products did not bind any GST constructs. With higher E7 concentrations, all pRB products bound the full length GST-E7 and CR3 domain fusion protein but did not bind GST. Complete binding was not observed, suggesting that denaturation, phosphorylation or other modifications render some fraction of these proteins unable to bind. The E7 CR2 and CR3 domains bind to non-overlapping domains on pRB. (0002)

Notes: [35S]methionine-labeled pRB60, pRB50 and pRB105 proteins were synthesized using TNT® Coupled Reticulocyte Lysate System. Experiments were performed with these proteins and GST fusion proteins to determine protein-binding sites. At low levels of GST-fusion protein, the pRB60 protein bound immobilized GST-E7(3-98) protein but did not bind the GST-E7-CR3 fusion or GST. The other truncated pRB translation products did not bind any GST constructs. With higher E7 concentrations, all pRB products bound the full length GST-E7 and CR3 domain fusion protein but did not bind GST. Complete binding was not observed, suggesting that denaturation, phosphorylation or other modifications render some fraction of these proteins unable to bind. The E7 CR2 and CR3 domains bind to non-overlapping domains on pRB. (1810)

Notes: [³⁵S]methionine-labeled RalGDS (guanine nucleotide dissociation stimulator) was transcribed and translated in the TNT^® T7 Coupled Reticulocyte Lysate System. The lysate was incubated with GTPase samples and used in immunoprecipitation assays with anti-RalGDS antisera. GST-R-ras, GST-H-ras and GST-Rap (Ras-like GTPases) bind with full length RalGDS in a GTP-dependent manner. In addition, Raf and RalGDS compete for binding to Ras-like GTPases. (1814)

Notes: p50, p65 and c-Rel (NF-kappaB/Rel proteins) were translated using the TNT® T7 Coupled Reticulocyte Lysate System. The proteins were checked for binding to NF-kappaB and Igkappa sites in electrophoretic mobility shift assays. p65 and c-Rel bind to TFkappaB-like sites, but neither p50/p65 heterodimers nor p50 homodimers bind to TFkappaB-like sites. p65, p65/p50 heterodimers and p50 homodimers all bind Igkappa but c-Rel does not bind Igkappa. (1843)

Notes: [³⁵S]labeled TBPAS proteins were generated by in vitro transcription/translation using the TNT^® Coupled Reticulocyte Lysate System. These proteins were incubated with hTAFII250 from S9 cells and whole cell extracts. In addition, TBPAS was incubated with GST-TFIIB: Approximately 10-fold more TBPAS was bound to the GST-TFIIB than to the GST alone. The results suggest that the recruitment of TBP into TFIID involves multiple interactions. (1783)

Notes: This paper discusses the use of the TNT^® Coupled Reticulocyte Lysate System to express mitochondrial genes in the mammalian cytosol and check for protein expression. (0324)

Notes: A 2kb fragment of the APC exon 15 (a mutation cluster region) was generated by PCR amplification from patient and tumor DNA. The PCR products were transcribed and translated in the TNT^® T7 Coupled Reticulocyte Lysate System and subjected to the protein truncation test (PTT), the PTT revealed that several truncation mutations in exon 15 were responsible for different familial adenomatous polyposis cases. (0216)

Notes: [³⁵S]methionine-labeled pp50 phosphoprotein was synthesized using the TNT^® T7 Coupled Reticulocyte Lysate System. The protein was used in DNA-binding studies and shown to bind to both ssDNA-cellulose and dsDNA-cellulose but did not bind unmodified cellulose. The results suggest that pp50 may have a higher affinity for ssDNA than for dsDNA. (1858)

Notes: EBNA2 and RBP-Jkappa were produced using a TNT® System. RBP-Jkappa specifically binds to the Cp and Tp EBNA2-responsive elements. EBNA2 and RBP-Jkappa coimmunoprecipitate when translated together. Also, EBNA-RBP-Jkappa -DNA complexes form in vitro with Cp or Tp oligonucleotides. (1860)

Notes: The membrane topology of the alpha subunit of the H+, K+-ATPase was investigated. DNA sequences encoding membrane-spanning segments were transcribed and translated using a troponin TNT^® Coupled Reticulocyte Lysate System in the presence of [³⁵S]methionine with and without microsomes. The data verify the first four and the eighth membrane-spanning segments of the alpha subunit. (2048)

Notes: [³⁵S]methionine-labeled Oct-1, Tef-1, Sp1 and TBP (cellular transcription factors) were synthesized using the TNT^® Coupled Reticulocyte Lysate System. The proteins were assayed for binding to GST fusion proteins, GST-IEP55 (immediate early protein 55kDa protein) and GST-IE86 or GST alone. Both GST-IEP55 and GST-IEP86 bind to TBP and Tef-1 but do not bind to Oct-1; GST-IEP86 also binds to Sp1. The results suggest that IEP86 activates transcription through interactions with the basal transcription complex and upstream bound factors. (1769)

Notes: RelA and NFKappaB1, tagged with HA, were synthesized from PCR products using the TNT® T7 Coupled Reticulocyte Lysate System. The proteins were coimmunoprecipitated with anti-HA and analyzed by SDS-PAGE. Multimerization was demonstrated for the proteins with the formation of the heterodimer being stronger than the homodimers. The proteins were also tested in gel mobility shift assays with kappaB DNA probes to demonstrate DNA binding specificity. (1830)