4.13. CREB Activation Assay

CREB transcription factor activation was measured using the pCREB-Luc CREB luciferase reporter vector (Signosis, Santa Clara, CA, USA). The cells were seeded at the density of 60,000 per well of 96-well plate the day before transfection and were cotransfected with the vector and the red fluorescent protein expression vector pTagRFP-N (Evrogen, Moscow, Russia) at a 1:1 ratio using the FuGENE HD transfection reagent (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The day after the transfection the cells were treated with the test substances for 3 h in a fresh medium, after which the RFP fluorescence was measured, the cells were lysed and used for the luciferase activity detection.