serum response element (sre-luc, pgl3.4,  (Promega)

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

Journal: The Journal of Neuroscience

Article Title: β-Amyloid Peptide at Sublethal Concentrations Downregulates Brain-Derived Neurotrophic Factor Functions in Cultured Cortical Neurons

doi: 10.1523/JNEUROSCI.5463-03.2004

Figure Lengend Snippet: Pretreatment with Aβ1-42 at sublethal concentrations decreased the level of BDNF-activated transcription factor Elk-1 (P-Elk-1) and the transcriptional activity of a serum response element-containing (SRE) construct. A, The phosphorylation of Elk-1 was examined by the use of an antibody against phosphorylated Elk-1 (P-Elk). Pretreatment with 5 or 10 μm Aβ1-42 for 2 hr attenuated the BDNF-induced increase in P-Elk-1 but had no significant effect on the total amount of Elk-1 (T-Elk). B, Quantification of the effects of pretreatment with 5 or 10 μm Aβ1-42 (B + A5 or B + A10). Estimates are the mean ± SEM (n = 3) expressed in terms of P-Elk-1 levels induced by BDNF (B). Effects of 5 and 10 μm Aβ1-42 (A5, A10) were significant (*p < 0.05, unpaired Student's t test). C, Analysis of SRE-mediated transcriptional activity. Cortical neurons at 3 DIV were transfected with plasmid pSRE-Luc containing SRE sequences and a luciferase reporter gene (see Materials and Methods). Cells transfected with a CMV-luciferase plasmid served to normalize SRE activity. After 40 hr the cultures were switched to fresh medium and incubated for 1 hr in the presence or the absence of 5 μm Aβ1-42. Transfected cortical cultures were incubated for an additional 9 hr either with or without the addition of 50 ng/ml BDNF, and transcriptional activity was measured by the luciferase assay. Aβ1-42 treatment decreased the BDNF-induced transcriptional activity of SRE. Estimates are the mean ± SEM (n = 3) expressed in terms of BDNF-induced transcriptional activity. The effect of Aβ1-42 was significant (*p < 0.05, unpaired Student's t test).

Article Snippet: Cortical neurons were transfected with the plasmid pCRE-Luc containing the cAMP response element (CRE) sequence (TGACGTCA) and pSRE-Luc containing the serum response element (SRE) sequence (CCATATTAGG) (Stratagene, La Jolla, CA) at 3 DIV with a procedure described by Myers et al. ( 1998 ).

Techniques: Activity Assay, Construct, Transfection, Plasmid Preparation, Luciferase, Incubation