8 pcpt cgmp

The anti-human/mouse RGS5 antibody was purchased from Sigma-Aldrich (GW22900, Schnelldorf, Germany). Its specificity on tissue samples was confirmed by comparing the RGS5 staining of capillary pericytes in muscle tissue of wild-type and RGS5-deficient mice (Supplementary Fig S15). The monoclonal anti-mouse CD31 antibody (clone: MEC 13.3) was obtained from Santa Cruz Biotechnology (Heidelberg, Germany). The anti-mouse αSMA antibody was purchased from Dianova (Hamburg, Germany), the anti-mouse PCNA antibody was ordered from Abcam (Cambridge, UK), the anti-mouse myocardin antibody was purchased from Santa Cruz (Heidelberg, Germany), and the anti-active-RhoA (Rho-GTP) antibody (#26904) was ordered from New East Biosciences (Pennsylvania, USA). TRITC-labelled phalloidin was obtained from Invitrogen. Bradykinin, angiotensin II, norepinephrine and NONOate were purchased from Sigma-Aldrich, sphingosine-1-phosphate was obtained from Cayman Chemical (New Orleans, USA) and 8pCPT-cGMP from BioLog (Bremen, Germany). The adenoviral RGS5, GFP and Lsc vectors were kindly provided by Prof. Wieland (Department of Experimental Pharmacology, Heidelberg University).

To identify palmitoylated proteins F11 cells were stimulated with 1 mM of 8-pCPT-cGMP (Biolog, #C 009-10) for 10 min followed by subcellular fractionation. F11 cells were prepared by homogenization in 0.34 M sucrose supplemented with protease blockers [aprotinin (20 U/μl, Carl Roth, #A162), leupeptin (5 mM, Sigma, #L2884), pepstatin A (5 mM, Sigma, #P5318), PMSF (1 mM, Sigma, #P7626)]. Nuclei were pelleted at 200 × g for 10 min and the resulting supernatant was centrifuged at 100,000 × g for 10 min to obtain a crude membrane pellet and the cytoplasmic fraction in the supernatant. The membrane fraction was solubilized in 1% CHAPS (Merck, #1116620010) in PBS supplemented with protease blockers. Un-solubilized material was removed by centrifugation at 100,000 × g for 10 min. Palmitoylated proteins were then labeled by the acyl-biotin exchange (ABE) chemistry protocol as described by Wan et al. (2007) (link). Free thiols were blocked by NEM (Thermo Scientific, #23030). Then thioester bonds between cysteines and palmitate were specifically cleaved by hydroxylamine treatment and finally newly free thiols were labeled by HPDP-biotin (Thermo Scientific, #21341) (see schematic in Figure 6D). Palmitoylated proteins were purified by streptavidin agarose pulldown (Thermo Scientific, #434341).

The following drugs were used: ANP and CNP (Tocris, Bristol, UK), DEA/NO (Axxora, Ann Arbor, MI, USA), glutamate (Sigma, St. Louis, MO, USA), IBMX (Sigma), Vinpocetine (Cayman, Ann Arbor, MI, USA), Bay 60-7550 (Cayman), EHNA (Axxora), Milrinone (Sigma), Sildenafil (Cayman), Zaprinast (Santa Cruz, Dallas, TX, USA), 8-Br-cGMP (Biolog, Bremen, Germany), and 8-pCPT-cGMP (Biolog).