Kt5823

Apocynin was purchased from SAFC (Saint. Louis, MO, USA). 8-Bromoguanosine 3′,5′-cyclic monophosphate, bradykinin, calcium ionophore (A23187), N (G)-nitro-L- arginine methyl ester (L-NAME), ODQ, tetrahydrobiopterin, siRNA against eNOS and N-TER Nanoparticle siRNA Transfection system, and anti-α-tubulin antibody were obtained from Sigma (Saint Louis, MO, USA). Anti-Shc/p66 (pSer36) antibody was purchased from Calbiochem (Darmstadt, Germany). DETA NONOate, DEA NONOate, and KT5823 were purchased from Cayman (Michigan, USA). Oxidized LDL and LDL are bought from Biomedical Technologies (Stoughton, MA, USA). Anti-Shc antibody was purchased from Cell Signaling (Danvers, MA, USA). Anti-eNOS antibody was bought from B&D transduction laboratories (NJ, USA). Anti-rabbit and Anti-mouse second antibodies were bought from GE healthcare (Buckinghamshire, UK).

Aβ1–42 was obtained from rPeptide. Bay 60-7550 (CAS: 439083-90-6) was obtained from Sigma-Aldrich. The mice were treated with different doses of Bay 60-7550 (0.5, 1.0, 3.0 mg/kg/day, i.p.) or vehicle for 14 days after microinjection of Aβ1–42. PKG inhibitor KT5823 (Cayman Chemical, United States) and PKA inhibitor H89 (Sigma-Aldrich, United States) were microinjected bilaterally into the cerebroventricular, 30 min prior to treatment with Bay 60-7550. Antibodies against CRF, GR, p-CREB, CREB, and BDNF were obtained from Abcam (Cambridge, MA, United States). All secondary antibodies were obtained from Beyotime Biotechnology (Haimen, China).

The following pharmacological agents were used in this study. BAY 60–2770 (a gift from Bayer Pharma AG, Germany); K⁺ channel blocker, 5-HD (5-hydroxydecanoate-sodium salt, Enzo, NY, USA); potent and selective inhibitor of PKG, KT5823 (2,3,9,10,11,12-hexahydro-10R-methoxy-2,9-dimethyl-1-oxo-9S, Cayman Chemical, MI, USA); mitochondria uncoupler, CCCP (carbonyl cyanide 3-chlorophenylhydrazone, Sigma, St. Louis, MO, USA).

Pf-2545920 was purchased from Selleck Chemicals (cat# S2687); KT-5823 (cat# 10,010,965) and H-89 (cat# 10,010,556) from Cayman Chemical; human recombinant EGF protein solution from ThermoFisher Scientific (cat# PHG0311L). MCI-030 was synthesized and characterized for purity and chemical structure by the Drug Discovery Research Center at our institution, as described previously [25 (link)]. Antibodies are described in Table S1.

Aβ1-42 (rPeptide, USA) was dissolved in 0.9 % sterile saline, at a final concentration of 0.4 μg/μl, and incubated at 37°C for 4 days to obtain aggregated Aβ before microinfusion into cerebroventricle (Wang et al., 2012 (link)). Rolipram (Sigma-Aldrich, USA) was prepared by being dissolved in 0.9% sterile saline containing 1% dimethyl sulfoxide (DMSO). One and a half months after microinjection with Aβ1-42, the mice were treated with different doses of Rolipram (0.1, 0.5, 1.0 mg/kg/day, i.p.) or vehicle for 2 weeks. KT5823 (Cayman Chemical, USA), a selective inhibitor of cGMP-dependent protein kinase (PKG), and H89 (Sigm-Aldrich, USA), a cAMP-dependent protein kinase (PKA), were dissolved in artificial cerebrospinal fluid (ACSF) and were bilaterally microinjected into the intracerebroventricular, 30 min before treatment with Rolipram.
The primary antibodies of anti-CRFR1, anti-BDNF, and anti-GR were purchased from Abcam Biotechnology Company (Abcam, Cambridge, MA). The anti-pCREB and anti-CREB were purchased from Merck Milipore (Millipore,Billerica,MA,USA). All the secondary antibodies (anti-rabbit lgG) were purchased from MultiSciences Biotech Co., Ltd. (MultiSciences, Hangzhou, China). The CORT ELISA kit was purchased from Enzo Life Sciences (Enzo Life Sciences, USA).