Pgl4.54 luc2 tk vector

5DIV hippocampal neurons were cotransfected with pNL1.1 (Nluc) vector (Promega) containing the –309/+42 region of the Kcc2 mouse gene and pGL4.54 (luc2/TK) vector (Promega) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Then, 48 hours after transfection, cultures were briefly washed with PBS and lysed in Passive Lysis Buffer (Promega). Both Nluc and luc2 luciferase activities were measured using Nano-Glo Dual-Luciferase Assay System (Promega).
pNL1.1 (Nluc) vector was modified by introducing in its multiple cloning region the –309/+42 region of Kcc2 mouse gene containing the Egr4 consensus sequence as the only binding site for transcription factors (as described previously in refs. 46 (link), 47 (link)). Subloning procedures were performed by Bio-Fab Research srl (Rome).

3T3-L1 cells were seeded at a density of 1.5 × 10⁴ cells in a 24-well culture plate and, 24 h later, cells were transfected using ViaFect transfection reagent (Promega Corporation, Madison, WI, USA). The total amount of DNA used for transfection per well was 0.8 μg, including approx. 0.5 μg of pNL1.1[Nluc] (equimolar concentrations of empty pNL1.1[Nluc], pNL1.1[Nluc]_Chemerin_Full, pNL1.1[Nluc]_Chemerin_Proximal, or pNL1.1[Nluc]_Chemerin_Distal) and 0.3 μg of the pGL4.54 [luc2/TK] vector (Promega Corporation, Madison, WI, USA) expressing the Firefly luciferase that was used as an internal control. Twenty-four hours after transfection, a portion of the cells was stimulated with IL-1β (10 ng/mL) and OSM (50 ng/mL) for 48 h. For methylation studies, cells were transfected with 0.5 μg of ligation reactions from the methylated/mock-methylated pNL1.1[Nluc]/chemerin promoter constructs and 0.3 μg of the pGL4.54 [luc2/TK] vector. The amount of DNA per well was equalized using mock plasmid DNA (pcDNA3.1; Promega). Forty-eight hours later, the cells were harvested by scraping into a passive lysis buffer (Promega Corporation, Madison, WI, USA). NanoLuc and Firefly activities in cell lysates were measured using the Nano-Glo Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA) according to the manufacturer’s protocols.