8 bromo camp

Primary mouse hepatocytes were cultured in William’s medium E (Thermo Fisher Scientific) supplemented with 10% foetal bovine serum at 37 °C in 5% CO₂. Cells were treated with either an adenovirus (ViraPower Adenoviral Gateway Expression Kit, Thermo Fisher Scientific) expressing mouse Nrg1α or siRNA (Supplemental Table 1, Integrated DNA Technologies KK, Tokyo, Japan) targeting for mouse Nrg1 24 h before stimulation with 500 μM 8-bromo-cAMP (Cayman Chemical, Ann Arbor, MI, USA) and 100 nM dexamethasone (Sigma) for 3 h. Viruses encoding β-galactosidase were used as a control. A multiplicity of infection of 50 for each virus was used for the infection. In some experiments, cells were pretreated with rNRG1α (1 or 10 nM) 30 min, or inhibitors such as Ly294002 (Wako Pure Chemical Industries), PD98059 (Cayman Chemical) or lapatinib (BioVision, Inc, Milpitas, CA, USA) 1 h before the stimulation. The conditioned media (24 h incubation) of isolated hepatocytes were collected to determine whether the N-terminal region of NRG1 endogenously expressed in hepatocytes was cleaved and released.

3-Isobutyl-1-methylxanthine (IBMX), bovine serum albumin (BSA), NaHCO₃ and Progesterone were purchased from Sigma-Aldrich (St. Louis, MO, United States). 8-Bromo-cAMP (sodium salt) and cAMP Elisa kit from Cayman Chemical (Ann Arbor, MI, United States). β-mercaptoethanol from Thermo Fisher Scientific (Waltham, MA, United States). PBS buffer was purchased from Corning (Radnor, PA, United States). Rabbit monoclonal anti-phospho-PKA substrates (anti-pPKA substrates) (clone 100G7E) and Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG, were purchased from Cell Signaling Technology (Danvers, MA). Mouse anti-phospho Tyrosine (anti-pTyr) (clone 4G10) was from EMD Millipore.