 Figure 1 - Expression kinetics from a synthetic model system for ERK signal duration. A: HEK293 with a stably transfected ∆RAF1:ER fusion protein were treated with ER antagonist 4-hydroxytamoxifen (4OHT, ON scenario in B). To generate pulses, ERK signalling was turned off using the MEK inhibitor U0126 (ON/OFF scenario in B). To distinguish between primary and secondary response genes, translation was blocked with cycloheximide (CYHX) in parallel to 4OHT stimulation (ON/CYHX scenario in B). In addition, we used actinomycinD (ActD) to determine mRNA half-lives via transcriptional shut-down and 4-thiouridine (4SU) to determine mRNA half-lives via metabolic labelling. 