serum response elements  (Addgene inc)

Journal: bioRxiv

Article Title: Is FAM19A5 an adipokine? Peripheral FAM19A5 in wild-type, FAM19A5 knock-out, and LacZ knock-in mice

doi: 10.1101/2020.02.19.955351

Figure Lengend Snippet: (A) An SRE-luc reporter assay was used to determine S1PR2 receptor activation in response to S1P and FAM19A5. SRE-luc reporter and S1PR2 genes were co-transfected into HEK293 cells that stably expressed Gqi protein. Cells in serum-free medium were then treated with both S1P and FAM19A5 for 6 h and subjected to a luciferase assay system. (B) Nano-luc reporter assay for β-arrestin recruitment to S1PR2. S1PR2-LgBit and β-arrestin-SmBit constructs were co-transfected into HEK293 cells. Under a live cell system, luminescence was determined before and after treatment with S1P and FAM19A5. Data are presented as the mean ± standard error of the mean from three independent experiments. (C) Internalization of S1PR2 in response to S1P and FAM19A5. HEK293 cells were transfected with a S1PR2-GFP construct. Cells were cultured under serum-free conditions for 16 hr and then treated with S1P, naïve FAM19A5, and FAM19A5-Cy3 for 30 min. Cellular locations of S1PR2-GFP in the presence of the ligands were determined using a confocal microscope. Scale bar represents 10 μm.

Article Snippet: The SRE-luciferase (SRE-luc) vector containing a single copy of the serum response element (SRE: CCATATTAGG) conjugated to luciferase was purchased from Stratagene (La Jolla, CA, USA).

Techniques: Reporter Assay, Activation Assay, Transfection, Stable Transfection, Luciferase, Construct, Cell Culture, Microscopy