Signaling pathways were analyzed 48 h after transfection. Intracellular cAMP accumulation for the determination of Gs/Gi activation was analyzed using the AlphaLISA technology (PerkinElmer, Rodgau, Germany). Human α-MSH was purchased from Sigma-Aldrich (Taufkirchen, Germany). Forskolin was used to investigate Gi activity. Stimulation was performed for 45 min. Cell lysis (50 μL/well lysis buffer) and cAMP measurement was conducted as described elsewhere [10 (link)] and according to the manufacturer´s protocol (PerkinElmer).
NFAT, SRE, and SRF activity were determined in luciferase reporter gene assays. Cell lysis was performed with 50 μl/well of 1x passive lysis Buffer (Promega). Pathway activities were determined by luciferase activity according to the manufacturer’s protocol (Promega).
NFAT, SRE, and SRF activity were determined in luciferase reporter gene assays. Cell lysis was performed with 50 μl/well of 1x passive lysis Buffer (Promega). Pathway activities were determined by luciferase activity according to the manufacturer’s protocol (Promega).
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