Figure 6. A GRN governed by an AND gate of stimulus-responsive mRNA synthesis and half-life control.

(A) Mature and nascent mRNA expression fold changes in BMDMs stimulated with TNF (10 ng/ml) or LPS (100 ng/ml). The shown data are representative of three independent experiments. The same stimulation conditions are used in all subsequent experiments.

(B) mRNA half-life measurements using the synthesis inhibitor actinomycin D in BMDMs stimulated with TNF (black) or LPS (red) for 30 minutes. The underlying qRT-PCR data was representative of three independent experiments. All mRNA half-lives were calculated within a 50% standard error by exponential regression analysis.

(C) Phospho-p38 (Thr180/Tyr182) and phospho-ERK1/ERK2 (Thr202/Tyr204) revealed by immunoblot of whole cell lysates prepared from stimulated BMDMs. ERK1 and ERK2 immunoblots are shown as loading controls. This is representative of three independent experiments performed by different individuals.

(D) and (E) Phospho-TTP (arrowhead), TTP and α-Tubulin (loading control) were analyzed by immunoblotting of whole cell lysates prepared from stimulated BMDMs with and without a p38 inhibitor (2.5 μM, SB202190). This is representative of three independent experiments performed by different individuals.

(F) A schematic of the AND gate between NFκB-driven transcription and LPS-specific activation of a p38/ERK- and TTP-dependent mRNA stabilization pathway. This is referred to as mod.GRN 2S, and its inclusion results in model v3.

(G) mRNA half-life measurements as in (B) in wild-type and erk2^−/− BMDMs stimulated with LPS (red) for 30 minutes or in the presence of p38 inhibitor (green for wild-type and yellow for erk2^−/−). The shown data are representative of three independent experiments.

(H) Mature and nascent mRNA expression fold changes in wild-type or erk2^−/− BMDMs stimulated with LPS, in the absence (red) or presence of p38 inhibitor (green for wild-type and yellow for erk2^−/−). The shown data are representative of three independent experiments.