Enhancer Activation Assay for Sox4, Sox11, Il15, Mapk1

ChIP-seq enhancer peak regions assigned to Sox4, Sox11, Il15 and Mapk1 were amplified from mouse genomic DNA and cloned into pBV- Luc, a luciferase reporter plasmid with a minimal promoter and very low basal activity (24 (link)). These reporter plasmids were co-transfected with 3X-FLAG SOX4 or 3X-FLAG SOX11 expression plasmids (14 (link)) into HEK293 cells using Viromer Red reagent (Origene). pSV2bgal plasmid was used as a control for transfection efficiency. Cells were treated with 10ng/mL recombinant TNF (R&D systems) for the last 16h of the 36h transfection period. Luciferase and β-galactosidase activities were assayed using the Dual-light combined reporter gene assay system (Applied Biosystems) using Synergy H1 Multi-Mode Plate Reader (Biotek). Reporter activities were normalized for transfection efficiency and reported as fold change in luciferase activity. Assays were performed as triplicates.

Free full text: Click here

Jones K., Ramirez-Perez S., Niu S., Gangishetti U., Drissi H, & Bhattaram P. (2022). SOX4 and RELA Function as Transcriptional Partners to Regulate the Expression of TNF- Responsive Genes in Fibroblast-Like Synoviocytes. Frontiers in Immunology, 13, 789349.

Publication 2022

Viromer Red reagent recombinant TNF Dual-light combined reporter gene assay system Synergy H1 Multi-Mode Plate Reader

Assays Cells Chip seq Genomic Hek293 cells Il15 Light Luciferase Mapk1 Mouse Plasmid Reporter gene Sox11 Transfection β galactosidase

Corresponding Organization :

Other organizations : Emory University, Atlanta VA Medical Center

Top 5 similar protocols

Protocol cited in 1 other protocol

Variable analysis

independent variables

Overexpression of 3X-FLAG SOX4 or 3X-FLAG SOX11
Treatment with 10ng/mL recombinant TNF

dependent variables

Luciferase activity

control variables

Transfection efficiency (measured using pSV2bgal plasmid)

controls

Positive control: pBV-Luc reporter plasmid with minimal promoter and low basal activity
Negative control: Not explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!