Kt5720

The antibodies used were: mouse anti-FLAG (M1, Sigma); rabbit anti-APPL1 (Cell Signaling Technology); rabbit anti-EEA1 (Cell Signaling Technology); mouse anti-GAPDH (Millipore); rabbit anti-p42/44 ERK and phospho-p42/44 ERK (Cell Signaling Technology); rabbit anti-phosphoserine (Millipore); goat anti-rabbit and anti-mouse AlexaFluor 488, 555, 568, and 647 (Thermo Fisher); and goat anti-rabbit and anti-mouse horseradish peroxidase (HRP) (Thermo Fisher Scientific). The inhibitors/activators used were: Dyngo-4a (Abcam) at 30 μM (45 min pre-treatment), KT5720 (Abcam) at 10 μM (15 min pre-treatment), and 8-Br-cAMP (Sigma) at 0.5 mM (15 min pre-treatment). LH and follicle-stimulating hormone (FSH) (A.F. Parlow, National Hormone and Peptide Program, Harbor-UCLA Medical Center) were used at 10 nM, and isoproterenol (ISO; Sigma) was used at 10 μM. All concentrations of ligands used (LH and FSH at 10 nM and isoproterenol at 10 μM) induce maximal cAMP responses from dose-response curves published previously (Bouvier et al., 1987 (link), Gudermann et al., 1992 (link), Alvarez et al., 1999 (link)).

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.