Journal:

Article Title: Induction of Bcl-x L Expression by Human T-Cell Leukemia Virus Type 1 Tax through NF-?B in Apoptosis-Resistant T-Cell Transfectants with Tax

doi:

Figure Lengend Snippet: Transactivation of bcl-x promoter by Tax. Jurkat cells (106) were cotransfected with 2 μg of the Luc reporter plasmid pGL2-3.2 driven by the bcl-x promoter, 1 μg of pSV–β-galactosidase, and 2 μg of Tax-expressing plasmids or its control vector pHβAPr-1-neo (βA). The Tax-expressing plasmids utilized were pβMT-2Tax (WT), pβTax703 (703), and pβTaxM22 (M22). Luciferase activities were normalized with β-galactosidase activity. Data represent the mean ± the standard deviation (SD) of three independent experiments.

Article Snippet: We also used the mouse bcl-x promoter-driven luciferase (Luc) reporter plasmid pGL2-3.2, which contained a 3.2-kbp fragment upstream of the bcl-x gene ( 19 ). pGL2(1160), pGL2(848), pGL2(822), and pGL2(631) were newly generated by insertion of DNA fragments of the bcl-x promoter at the base positions of −1160 to −75, −848 to −75, −822 to −75, and −631 to −75 from the translation initiation ATG of the bcl-x gene, respectively, into the basic pGL2 vector containing a Luc reporter gene (Promega, Madison, Wis.).

Techniques: Plasmid Preparation, Expressing, Luciferase, Activity Assay, Standard Deviation

Journal:

Article Title: Induction of Bcl-x L Expression by Human T-Cell Leukemia Virus Type 1 Tax through NF-?B in Apoptosis-Resistant T-Cell Transfectants with Tax

doi:

Figure Lengend Snippet: Mapping of HTLV-1 Tax-responsive region in the bcl-x promoter. A series of Luc reporter plasmids based on pGL2 vectors containing fragments of 5′ flanking region of the bcl-x gene were cotransfected into Jurkat cells with WT Tax plasmid pβMT-2Tax (solid bars) or its control plasmid (open bars), together with reference plasmid pSV–β-galactosidase. Luciferase activities normalized with β-galactosidase activities after 48 h of incubation are indicated. Constructs of the reporter plasmids utilized are schematically illustrated in the left panel. Fragments of the 5′ flanking region of bcl-x gene are represented by heavy lines, and the number at the 5′ end of each construct indicate the 5′-most end base positions from the translation initiation codon of the bcl-x gene. The locations of the binding sites for transcription factors NF-κB (□), SP-1 (private-char pc1), Ets-1 (private-char pc1) and GATA-1 (private-char pc1), identified by using a transcription factor database (1a), are also indicated. pGL2κBM contained mutations within the NF-κB site (⊠) as described in Materials and Methods. Similar results were obtained in two independent experiments.

Article Snippet: We also used the mouse bcl-x promoter-driven luciferase (Luc) reporter plasmid pGL2-3.2, which contained a 3.2-kbp fragment upstream of the bcl-x gene ( 19 ). pGL2(1160), pGL2(848), pGL2(822), and pGL2(631) were newly generated by insertion of DNA fragments of the bcl-x promoter at the base positions of −1160 to −75, −848 to −75, −822 to −75, and −631 to −75 from the translation initiation ATG of the bcl-x gene, respectively, into the basic pGL2 vector containing a Luc reporter gene (Promega, Madison, Wis.).

Techniques: Plasmid Preparation, Luciferase, Incubation, Construct, Binding Assay

Journal:

Article Title: Induction of Bcl-x L Expression by Human T-Cell Leukemia Virus Type 1 Tax through NF-?B in Apoptosis-Resistant T-Cell Transfectants with Tax

doi:

Figure Lengend Snippet: Involvement of NF-κB pathway in Tax-induced transactivation of bcl-x promoter. (A) Jurkat cells were cotransfected with 4 μg of bcl-xl promoter–Luc reporter plasmid pGL2-3.2, 1 μg of pSV–β-galactosidase, and various combinations of 2 μg of pβMT-2Tax and 2 μg of pcDNA3-IκBαM as indicated. The total plasmid amount for transfection was always adjusted up to 9 μg by adding control plasmids pHβAPr-1-neo or pcDNA3. The Luc activity of the samples after 48 h of incubation was normalized to the β-galactosidase activity. Data represent the mean ± the SD of three independent experiments. (B) Control experiments with reporter plasmids without potential NF-κB binding sites. Jurkat cells were transfected with 4 μg of HTLV-LTR (21 bp)-Luc reporter plasmid WT-Luc, 2 μg of pβMT-2Tax, 1 μg of pSV–β-galactosidase, and 2 μg of pcDNA3-IκBαM or pcDNA3. The luciferase activity was indicated as described in panel A.

Article Snippet: We also used the mouse bcl-x promoter-driven luciferase (Luc) reporter plasmid pGL2-3.2, which contained a 3.2-kbp fragment upstream of the bcl-x gene ( 19 ). pGL2(1160), pGL2(848), pGL2(822), and pGL2(631) were newly generated by insertion of DNA fragments of the bcl-x promoter at the base positions of −1160 to −75, −848 to −75, −822 to −75, and −631 to −75 from the translation initiation ATG of the bcl-x gene, respectively, into the basic pGL2 vector containing a Luc reporter gene (Promega, Madison, Wis.).

Techniques: Plasmid Preparation, Transfection, Activity Assay, Incubation, Binding Assay, Luciferase

Journal:

Article Title: Induction of Bcl-x L Expression by Human T-Cell Leukemia Virus Type 1 Tax through NF-?B in Apoptosis-Resistant T-Cell Transfectants with Tax

doi:

Figure Lengend Snippet: Inhibition of constitutive activation of bcl-xl promoter in CTLL-2/Tax transfectants by IκBαM. (A) CTLL-2/WT clone cells were transfected with pGL2-3.2, pSV–β-galactosidase, and either pcDNA3-IκBαM (solid bar) or pcDNA3 (open bar). Luc activity after 48 h of incubation in the absence of IL-2 was normalized to β-galactosidase activity and indicated as the percentage of activity in the IκBαM(−) sample. Data represent the mean ± the SD of three independent experiments. (B) Control experiments with the WT-Luc reporter plasmid instead of pGL2-3.2.

Article Snippet: We also used the mouse bcl-x promoter-driven luciferase (Luc) reporter plasmid pGL2-3.2, which contained a 3.2-kbp fragment upstream of the bcl-x gene ( 19 ). pGL2(1160), pGL2(848), pGL2(822), and pGL2(631) were newly generated by insertion of DNA fragments of the bcl-x promoter at the base positions of −1160 to −75, −848 to −75, −822 to −75, and −631 to −75 from the translation initiation ATG of the bcl-x gene, respectively, into the basic pGL2 vector containing a Luc reporter gene (Promega, Madison, Wis.).

Techniques: Inhibition, Activation Assay, Transfection, Activity Assay, Incubation, Plasmid Preparation