Figure 4
From: Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression
EPAC-1/2 activity is required for α-MSH-induced CREB phosphorylation.
In (A) Lysates of mHypoA-2/10-CRE cells stimulated with 1 μM of α-MSH for various periods of time were subjected to western-blot analysis using either a phospho-specific antibody against p-CREB (Ser-133) or against the total tubulin protein as a loading control. One representative blot is shown. Data of 12 independent experiments were quantified by densitometry and ratios between p-CREB and tubulin signals calculated. Asteriks indicate a significant difference to time point zero using one-way ANOVA followed by Dunnett’s analysis. In (B) lysates of cells preincubated with KT-5720 (5 μM) and in (C) with ESI-09 (20 μM) or with the carrier DMSO (0.1% or 0.2% respectively ESI-09) for 30 min and then stimulated with 1 μM α-MSH for 5 and 10 min were subjected to western-blot analysis using either a phospho-specific antibody against p-CREB (Ser-133) or against the total tubulin protein as a loading control. One representative blot is shown. Data of 6 (B) or 8 (C) independent experiments were quantified by densitometry, ratios between p-CREB and tubulin signals calculated, α-MSH-induced CREB phosphorylation normalized to not stimulated cells. Hashed signs indicate a significant difference to zero using the one-sample Student’s t-test. Asterisks indicate a significant difference between ESI-09- and DMSO-treated cells using the two-sample Student’s t-test.
