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Dual-Luciferase reporter assay was used to detect the transcriptional activity of the constructed promoter.
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Whereas the −980, −650 and −450 to +80 fragments of the EBP1 promoter were activated to similar extents by E2F1 or E2F3, transcriptional activity of the construct containing the region from −100 to +80 was not significantly altered (Fig 2B).
Firefly activity of experimental constructs was divided by the Firefly activity of the construct without IRES (pLL).
We then tested the biological activity of the construct.
This mechanism may explain the reduced transcriptional activity of the construct containing the rs3289 T allele.
Induction of miR-27a reduced the reporter activity of the construct containing wild type 3′-UTR of EGFR.
The relative luciferase activity of the construct with wild-type 3′UTR was significantly repressed following miR-223 transfection (P < 0.05).
Conversely, substituting either the bHLH or C-terminus of NeuroD into a chimera reduces the activity of the construct when compared to wild type Ngn2.
The knockdown of Jarid1b, but not that of Jarid1a (not shown), led to an increase in luciferase activity of the constructs containing minimal repressive region while it did not affect the activity of the construct which lacks the minimal repressive region.
The relative luciferase activity of the construct containing the DR-1 and DR-2 elements was significantly higher than that of the construct lacking these elements (* P < 0.01, Fig. 5b).
Inhibition of the Egr2 levels (by ∼75%) caused an increase in the luciferase activity of the reporter constructs containing the minimal repressive region: pGL3 (−3.3 to 0) and pGL3 (−3.3 to −1.9), while it did not affect the activity of the construct (−3.3 to −2.7) which lacks the minimal repressive region.
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