P pka substrate

Soleus muscle samples were extracted using radioimmunoprecipitation assay buffer (Tris-HCl, pH 8.0, 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, protease inhibitor cocktail, and phosphatase inhibitor cocktail). The proteins were resolved by 10 or 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked overnight with 5% skim milk in PBST (8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, 0.24 g KH₂PO₄, pH 7.4, 1% Tween-20) and then incubated for more than 2 h with primary antibody (PKA; sc-98,951, Santa Cruz Biotechnology, Dallas, TX, USA, Plin5; sc-240,627, Santa Cruz Biotechnology, p-PKA substrate; 9621 s, Cell Signaling Technology, Danvers, MA, USA, CGI-58; sc-100,468, Santa Cruz Biotechnology, ATGL; sc-67,355, Santa Cruz Biotechnology, HSL; sc-25,843, Santa Cruz Biotechnology). Membranes were developed using horseradish peroxidase-conjugated anti-goat, mouse, or rabbit IgG, followed by incubation with ECL solution (Amersham Pharmacia Biotech, Piscataway, NJ, USA), followed by detection using a Fuji LAS-4000 Imaging station (ImageQuantTMLAS-4000, GE Healthcare, Little Chalfont, UK).

PGI₂ (Cayman Chemical, Michigan, USA), Forskolin (Sigma-Aldrich, UK), Fibrinogen (Enzyme Research, Swansea, UK), Y-27632 (Abcam, Cambridge, UK), Rp-8CPT-cAMP (Biolog, Bremen, Germany), KT5720 (Abcam, Cambridge, UK), pRhoA^ser188 (Santa-Cruz Biotechnology, Heidelberg, Germany), pMLC^ser19 and pVASP^ser157 (New England Biolabs, Hitchin, UK), GAPDH and Arp2/3 (Millipore, Watford, Hertfordshire, UK), PKA RI (Cell Signalling Technology, Leiden, Netherlands), PKARII (BD Biosciences, Oxford, UK), pPKA substrate (Cell Signalling Technology, Leiden, Netherlands), WASP (Santa-Cruz Biotechnology, Heidelberg, Germany), pTyrosine (Cytoskeleton, Denver, UK), RhoA pulldown kit (Cytoskeleton, Denver, UK), cAMP assay and ProLong Diamond Antifade Mountant (GE healthcare, Little Chalfont, Buckinghamshire, UK), Flourescent secondary anti-mouse 800 and anti-rabbit 680 antibodies (LI-COR Biotechnology, Cambridge, UK) All other chemicals were from Sigma Ltd (Poole, UK) unless otherwise stated.

Total proteins were extracted from lung tissues using RIPA buffer containing proteinase inhibitor. Protein concentrations were determined by the BCA protein assay method. The protein samples were separated on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred to the polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline and Tween 20 (TBS-T) for 1 h at room temperature. Then membranes were incubated overnight with the primary antibody at 4 °C. Antibodies against VPAC2 (1:1000; Affinity, Cincinnati, OH), GATA3, cAMP (1:1000, Proteintech, Wuhan, China), p-PKA substrate and β-actin (1:1000, Cell Signaling Technology, Danvers, MA) were used. The membranes were washed in TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Immunoreactive bands were visualized by ECL detection (Millipore, Temecula, CA). Image J software (NIH, Bethesda, MD) was used to quantify the protein levels. β-actin was used for standardization.