Human α msh

Signaling pathways were analyzed 48 h after transfection. Intracellular cAMP accumulation for the determination of Gs activation was analyzed using the AlphaLISA technology (PerkinElmer, Rodgau, Germany). Human α-MSH was purchased from Sigma-Aldrich (Taufkirchen, Germany). Stimulation was performed for 45 min. Cell lysis (50 μl/well lysis buffer) and cAMP measurement were conducted as described elsewhere (22 (link)) according to the manufacturer’s protocol (PerkinElmer).
NFAT, SRE, and NFκB activity were determined in luciferase reporter gene assays. Stimulations with α-MSH, TNFα, and Ghrelin (Sigma-Aldrich) were performed for 6 h to allow appropriate reporter expression and according to the manufacturer’s instructions (Promega). Subsequently, cell lysis was performed with 50 μl/well of 1× Passive Lysis Buffer (Promega). Pathway activities were determined by luciferase activity according to the manufacturer’s protocol (Promega).

Nle(4),D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH) was from Phoenix Pharmaceuticals, Inc. (Cat. no. 043-06, Burlingame, CA, USA). HS014 was from Tocris (Cat. no. 1831, Abingdon, UK). Human α-MSH, D-glucose and bombesin were from Sigma Aldrich (Cat. no. M4135, G8270 and B4272, Brøndby, Denmark).

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).