Notes: Biotinylated dsDNA containing the NFI binding site was incubated with the TNT^® Coupled Reticulocyte Lysate System. Streptavidin-coated beads were added, and the DNA-protein complexes and beads were removed through magnetic fractionation. The supernatant was then used as the lysate without endogenous DNA-binding proteins present. (1823)

Notes: V. californica VAMP was translated in vitro using the TNT® T7 Coupled System. It bound VAP-33 and GST-syntaxin fusion protein specifically. This result suggests that additional factors may not be required for binding to occur. (1812)

Notes: [³⁵S]Cysteine-labeled beta-myb was synthesized using the TNT^® T3 Coupled Reticulocyte Lysate System. The protein was the approximate size predicted from the cDNA. (1449)

Notes: IRF-1 and IRF-2 (interferon (IFN) regulatory factors 1 and 2), the positive and negative regulators of IFN-beta transcription, were produced using the TNT^® Coupled Reticulocyte Lysate System. The proteins were analyzed in EMSAs with wild type and mutant CCE (cell-cycle element) probe. Both IRF-1 and IRF-2 bind CCE specifically. (1819)

Notes: Estrogen receptor knockout mice (ERKO) were created in previous work. Estrogen receptor (ER) from the ERKO line was in vitro translated using the TNT^® Coupled Reticulocyte Lysate System using [³⁵S]labeled methionine. The ERKO mice have a smaller ER that has reduced estrogen-dependent transcriptional activity compared to wild type. (1283)

Notes: [³⁵S]methionine-labeled ApCREB2 and ApC/EBP were translated in the TNT^® Coupled Reticulocyte Lysate System. In vitro interaction assays were performed with the labeled ApCREB2 and ApC/EBP and with GST fusions of ApCREB2 and its deletion mutants. ApCREB2 forms weak homodimers, and ApCREB2 interacts with ApC/EBP. (1781)

Notes: [³⁵S]methionine-labeled Ets-1 was synthesized in vitro using the TNT^® Coupled Reticulocyte Lysate System. In vitro translated [³⁵S]labeled Ets-1 was incubated with immobilized ATF-2194 on nitrocellulose membranes and with GST-ATF-2194 on glutathione-agarose beads, and the proteins were shown to associate. (1793)

Notes: The HSV-1 protease UL26-encoded protein was produced using the TNT^® Coupled Reticulocyte Lysate System. The N-terminal autoproteolysis of UL26 protein was shown in vitro and was abolished when residues 245-248 were deleted. The autoproteolysis of this protease may be achieved in a defined order. (1107)

Notes: Bik was identified, cloned, and characterized. Bik contains a short sequence with homology to Bax and Bak that may be sufficient for protein interaction. [³⁵S]methionine-labeled proteins were expressed in vitro using the TNT^® Coupled Reticulocyte Lysate System. The Bik, Bax, and Bak proteins were tested for interaction with GST-Bcl-2, GST-Bcl-xL or GST. Bik interacts with Bcl-2 and Bcl-xL in vitro. (1750)

Notes: RBP and TFIIB were synthesized using the TNT® Coupled Reticulocyte Lysate System and Trans[35S]label (ICN). These proteins were used in in vitro protein association assays in the presence of DNA containing E2 and TBP binding sites. Cooperative DNA binding by E2 and TBP is mediated by the direct binding of E2 to TBP. (1832)

Notes: [³⁵S]labeled CREB or mutated CREB proteins were synthesized in the in vitro TNT^® Coupled Reticulocyte Lysate System. These proteins were mixed with ETFIIB and coimmunoprecipitated with anti-T7 Tag epitope mAb. (1780)

Notes: CDEBP was synthesized using the TNT^® T7 Coupled Reticulocyte Lysate System. [³⁵S]-labeled protein was analyzed by SDS-PAGE and immunoprecipitated with anti-CDEBP polyclonal antibody. The unlabeled protein was shown to bind the CDEI motif in gel shift assays (EMSA). (2043)

Notes: The cDNA clone of haPPARγ was transcribed and translated in the TNT^® Coupled Reticulocyte Lysate System. The protein product was labeled with [³⁵S]methionine and used in electrophoretic mobility shift assays (EMSAs) with [³²P]labeled dsDNA probe. The probe used was the PPRE (peroxisome proliferator response element) from the acyl-CoA oxidase promoter. The haPPARγ effectively bound PPRE. (1479)

Notes: The cDNA for type IV adenylyl cyclase (ACIV) was expressed in the TNT^® Coupled Reticulocyte Lysate System in the presence of [³⁵S]methionine. Adenylyl cyclase activity was assayed, and the protein was immunoprecipitated with antibodies to ACIV. The adenylyl cyclase was activated by bovine brain Gs and forskolin and was quite stable. This report showed that ACIV can be produced in vitro with the activity of baculovirus-produced ACIV. (1777)

Notes: Different RD1-CAT [³⁵S]methionine-labeled chimeric proteins were produced using the TNT^® Coupled Reticulocyte Lysate System. The chimeric proteins showed that the unique N-terminal domain of RD1 confers membrane association upon the cytosolic CAT protein. (0419)

Notes: [³⁵S]methionine-labeled wild type and mutant CREB proteins and CREB-Jun chimeras were synthesized using the TNT^® T7 Coupled Reticulocyte Lysate System. The dimerization of these proteins was analyzed in vitro. CREB proteins with double mutations in the leucine zipper region were unable to interact with Tax whereas CREB proteins with only single mutations in the region or wild type CREB proteins were able to dimerize with Tax. (GST analyses were performed with bacterial expressed proteins.) (1789)

Notes: [35S]methionine-labeled pre-IL-1beta and PARP(T) proteins were synthesized using the TNT® T7 Coupled Reticulocyte Lysate System and were used for cleavage studies. The proteins were incubated with purified recombinant human ICE, which cleaved both proteins. The cleavage of PARP requires 50- to 100-fold higher concentrations of ICE than required for pre-IL-1beta cleavage. (1904)

Notes: [³⁵S]methionine-labeled SCL (HLH transcription factor) and LYL-1 (a related bHLH protein) were produced using the TNT^® T7 Coupled Reticulocyte Lysate System. The proteins were run on SDS-PAGE before and after immunoprecipitation with anti-SCL antiserum. The anti-SCL immunoprecipitated the SCL protein but not the LYL-1 protein. (0633)

Notes: ELT-2 protein was produced using the TNT® System. The DNA binding activity was analyzed by gel mobility shift assays using the putative gut activator region of the C. elegans ges-1 gene. The protein binds the GATA sequences of the gene and is competed effectively with unlabeled oligo, except when those GATA sequences in the competitor are mutated to GTCGCC. (1802)

Notes: A yeast 2-hybrid interaction cloning system was used to isolate the novel interacting protein, FADD, which interacts with the cytoplasmic domain of Fas. Labeled FADD was prepared in vitro using the TNT^® T7 Coupled Reticulocyte Lysate System and used to demonstrate that the death domains of FADD and GST-Fas interact. (1187)

Notes: Pbx1, Pbx2, Hoxb-8, Hoxb-7, and Engrailed-2/Pbx1 fusion proteins were produced in the TNT^® SP6 Coupled Reticulocyte Lysate System. In vitro translated proteins were analyzed by EMSA using various DNA fragments. Hoxb-8 modulates the DNA binding activity of Pbx1 and Pbx2; Pbx1 and Hoxb-8 bind to DNA as heterodimers; Pbx1 and Pbx2 bind to DNA cooperatively with Hoxb-7. (1818)

Notes: Human adenovirus serotypes 3 and 5 were expressed in vitro using the TNT^® T7 Coupled Reticulocyte Lysate System and were labeled with [³⁵S]methionine. The proteins were then analyzed by SDS-PAGE and fluorography and by binding to HeLa cell monolayers followed by SDS-PAGE. The trimeric forms of the proteins were the only forms able to bind to the cells. Other work shows that the proteins bind different cellular receptors. (1788)

Notes: C-terminal truncated RAP74 proteins were synthesized in the TNT^® Coupled Reticulocyte Lysate System. These truncated mutants were used in coimmunoprecipitation assays with HA-hTAFII250. The recognition interfaces between the proteins were mapped using these assays. (1774)

Notes: The G22 gene from Lymantria dispar was cloned, sequenced and expressed in the TNT^® Coupled Reticulocyte Lysate System. The predicted size of the protein was in agreement with that of the in vitro generated protein. The protein has no significant sequence homology. (1415)

Notes: Peroxisome proliferator-activated receptor-alpha (PPARalpha) and retinoid-X receptor-alpha (RXRalpha) were synthesized using the TNT® Coupled Reticulocyte Lysate System. These proteins were used in gel retardation assays with two possible peroxisome proliferator response elements (PPREs). Element II is a functional PPRE, and both elements were responsive to activated RXRalpha. Neither RXRalpha nor PPARalpha were able to bind the elements alone, but as a heterodimer they were able to bind element II or both elements I and II. There was no binding by the heterodimer to element I in the absence of element II observed. (1825)