Journal: BMC Molecular Biology

Article Title: Identification of a candidate alternative promoter region of the human Bcl2L11 (Bim) gene

doi: 10.1186/1471-2199-9-56

Figure Lengend Snippet: BCL2L11(BIM)-P1 activity is regulated by E2F . For simplicity the alias BIM is used in the figure instead of the approved gene symbol BIM. A . Inducibility of endogenous E1 and E2-bearing BIM transcripts by transiently transfected E2F expression construct in HEK293 cells; real-time experiments were performed as previously in cells transfected with either pBst or an E2F expression construct, and the fold inducibility (relatively to pBst transfected cells) of individual transcripts by the E2F expression plasmid is presented; the results confirm reporter data and show that the expression of endogenous BIM transcripts derived from P1 can be induced by E2F. *p < 0.01, Anova: single factor. B . Analysis of the effects of E2F overerexpression on pBIM-P1-1918(+) activity in HEK293 cells. A concentration-dependent inducibility of P1 activity is observed; by contrast, a version of P1 comprising a 5' deletion of 1300 bp results in a promoter construct with a basal activity comparable to P1-1918, but without the capacity for E2F-mediated activation, suggesting the presence of E2F responsive elements in the deleted region. *p < 0.01, Anova: single factor.

Article Snippet: HEK293 cells were cotransfected with 0.05 μg of reporter plasmid, 0.01 μg of Renilla vector (pRL-TK, Promega), 0.01 μg of empty plasmid Bluescript (Stratagene, Inc.) or 0.1 or 0.05 μg Human E2F expression construct (Origene, Inc.) in 95 μl of serum-free medium per well in 96-well plate using Lipofectamine 2000 [ ].

Techniques: Activity Assay, Transfection, Expressing, Construct, Plasmid Preparation, Derivative Assay, Concentration Assay, Activation Assay

Journal: BMC Molecular Biology

Article Title: Identification of a candidate alternative promoter region of the human Bcl2L11 (Bim) gene

doi: 10.1186/1471-2199-9-56

Figure Lengend Snippet: Effects of E2F on BCL2L11-P1 constructs . For simplicity the alias BIM is used in the figure instead of the approved gene symbol BCL2L11. A . Sequence of the -1918 P1 region showing the position of 5' deletion variants. Forward primers used to generate the various P1 5' deletion variants are indicated (arrows). The putative E2F binding site is boxed, and the transcript initiation site is indicated (+1). B . Analysis of the effects of E2F over-expression on pBIM-P1 and 5'deletion variants activity in HEK293 cells. A concentration-dependent inducibility of P1 activity is observed in construct pBIM-P1-1918, -1565 and -1326, which contain a putative E2F responsive element. By contrast, pBIM-P1-1284 and -940 display basal activities comparable to pBCL2L11-P1-1918, but without the capacity for E2F-mediated response, suggesting a loss of E2F responsive element(s). *p < 0.05 and **p < 0.01, Anova: single factor.

Article Snippet: HEK293 cells were cotransfected with 0.05 μg of reporter plasmid, 0.01 μg of Renilla vector (pRL-TK, Promega), 0.01 μg of empty plasmid Bluescript (Stratagene, Inc.) or 0.1 or 0.05 μg Human E2F expression construct (Origene, Inc.) in 95 μl of serum-free medium per well in 96-well plate using Lipofectamine 2000 [ ].

Techniques: Construct, Sequencing, Binding Assay, Over Expression, Activity Assay, Concentration Assay

Journal: BMC Molecular Biology

Article Title: Identification of a candidate alternative promoter region of the human Bcl2L11 (Bim) gene

doi: 10.1186/1471-2199-9-56

Figure Lengend Snippet: E2F binding to a candidate E2F binding site in BCL2L11-P1 . EMSA on the candidate E2F binding site (CTCTGCGCGCCAGAGG). HEK293 cells were transfected with the E2F expression construct, and nuclear extracts were assayed for capacity for specific binding to the candidate E2F binding site through competition with a specific (E2F) or unspecific (NF-κB) unlabelled competitor double stranded (ds) oligonucleotide. A specific shift is identified (arrow) upon transfection with the E2F expression plasmid, which can only be competed with a specific ds oligonucleotide.

Article Snippet: HEK293 cells were cotransfected with 0.05 μg of reporter plasmid, 0.01 μg of Renilla vector (pRL-TK, Promega), 0.01 μg of empty plasmid Bluescript (Stratagene, Inc.) or 0.1 or 0.05 μg Human E2F expression construct (Origene, Inc.) in 95 μl of serum-free medium per well in 96-well plate using Lipofectamine 2000 [ ].

Techniques: Binding Assay, Transfection, Expressing, Construct, Plasmid Preparation