Journal: Molecular Biology of the Cell

Article Title: Down-regulation of the Wnt/β-catenin signaling pathway by Cacnb4

doi: 10.1091/mbc.E17-01-0076

Figure Lengend Snippet: Effect of β 4 -subunit on TCF and SRE-dependent transcription. (A) Luciferase activity measured in TCF + /Wnt3 + /FZD7 + , TCF + , and TCF − cells expressing eGFP (black bars) or β 4 -eGFP (gray bars). (B) copGFP fluorescence measured in TCF + /Wnt3 + /FZD7 + , TCF + , and TCF − cells expressing pcDNA (black bars) or β 4 -myc (gray bars). Luciferase and copGFP activity were normalized to the value measured in TCF + /Wnt3 + /FZD7 + cells expressing eGFP (A) or pcDNA (B). Experiments were done in triplicate with n = 3. (C) Luciferase activity measured in HCC cells expressing SRE reporter gene system, pTK-RL vector, and eGFP (black bars) or β 4 -eGFP (gray bars). SRE-dependent luciferase activity was normalized to Renilla luciferase activity ( n = 2). Transfected HCC cells were maintained in serum-free medium (− serum). Serum (10% final concentration) was added 4 h before luciferase measurement (+ serum). NS, nonsignificant; *, p ≤ 0.05; ****, p ≤ 0.0001.

Article Snippet: SRE-Luc reporter luciferase activity was normalized to the Renilla luciferase activity of the cotransfected control vector pTK-RL (Promega).

Techniques: Luciferase, Activity Assay, Expressing, Fluorescence, Plasmid Preparation, Transfection, Concentration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Novel Mechanism of Alternative Promoter and Splicing Regulates the Epitope Generation of Tumor Antigen CML66-L 1

doi:

Figure Lengend Snippet: Predominant expression of CML66-L in tumor cells resulted from tumor-specific transcription of CML66-L promoter. A, The schematic representation of CML66-L-specific promoter (1.8 kb) and CML66-S promoter (2.6 kb). Several transcription factor binding sites and their positions are marked on the CML66-L promoter and CML66-S promoter, respectively. B, Constructs of CML66-L promoter and CML66-S promoter in both sense and antisense orientations on the pGL-3 luciferase enhancer reporter vector. CML66-L promoter antisense and CML66-S promoter antisense were also constructed as controls, because it is well accepted that promoter activities are orientation dependent. C, CML66-L and CML66-S promoter activities in Hela cells. The mean ratios of firefly luciferase activities conferred by reporter constructs vs Renilla luciferase activities (internal control) and 2 SD are shown as relative luciferase activities for every experimental group. The error bars indicating the upper limit in all the groups, except CML66-L promoter sense orientation, are hardly visible due to the scale. The experiments were repeated four times, and the representative results are shown. Notably, similar results were obtained in HCCT, a hepatic carcinoma cell line, after transfection with the same sets of reporter luciferase vectors (not shown). D, CML66-L and CML66-S promoter activities in NTERA-2 cl.D1 human testicular embryonal carcinoma cells. The mean ratios of firefly luciferase activities conferred by reporter constructs vs Renilla luciferase activities (internal control) and 2 SD are shown as relative luciferase activities for every experimental group. The error bars indicating the upper limit in all the groups, except CML66-L promoter sense orientation construct and CML66-S promoter sense orientation construct, are hardly visible due to the scale. The experiments were repeated four times, and the representative results are shown.

Article Snippet: Cotransfected Renilla luciferase control vector (Promega) provides the expression of Renilla luciferase as an internal control.

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