Anti pcreb

Following stimulation, cells were washed with cold phosphate-buffered saline (PBS) and collected via scraping. Nuclear extracts were prepared using a NE-PER kit from Pierce (Rockford, IL). Protein concentration in the extracts was determined using the BCA protein assay kit (Pierce, Rockford, IL). The extracts were stored at −80 °C prior to use. Proteins (10 µg per lane) were separated by 12.5% SDS-PAGE and transferred to a nitrocellulose membrane. Western blotting was carried out using following primary antibodies according to manufacturer protocol: anti-CREB (06–863, Millipore, Billerica, MA), anti-pCREB (06–519, Millipore, Billerica, MA), anti-TORC1 (4E7-C1-F9-E6, GeneTex, Irvine, CA), anti-TORC2 (ab167129, Abcam, Cambridge, MA), anti-TORC3 (EPR3440, GeneTex, Irvine, CA), and anti-β-actin (#4967, Cell Signaling, Danvers, MA). Appropriate secondary antibodies conjugated with horseradish peroxidase were obtained from Jackson ImmunoResearch (West Grove, PA). Signals were visualized by chemiluminescence using ECL detection reagents (GE Healthcare, Piscataway, NJ), captured by ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA), quantified using ImageJ software [34] (link), and normalized to β-actin for comparison.

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).

Rats were deeply anesthetized with isoflurane and transcardially perfused with 4% paraformaldehyde prepared in phosphate buffer saline (PBS) buffer. Whole brain was collected and incubated in 4% paraformaldehyde for 16 h, transferred to 30% sucrose prepared in PBS, and stored at 4 °C for at least 48 h before tissue processing. Each brain was sectioned in 40-µm-thick coronal slices. Sections containing striatum were stained with anti-Iba1 (Wako: 019-19741; diluted 1:500), anti-glial fibrillary acidic protein (GFAP; Cell Signaling Technology: #3670; diluted 1:300), or anti-pCREB (Millipore: 06-519; diluted 1:500) antibodies.
Sections were incubated with secondary antibody goat anti-rabbit conjugated to Alexa Fluor 594 (Iba1) and goat anti-mouse conjugated to Alexa 488 (GFAP and pCREB), both diluted 1:200 in blocking buffer (0.3% Goat serum, 0.3% Triton X-100, 0.2% Tween, 10% 10X PBS in water). Sections were imaged on a Zeiss fluorescence confocal microscope with a 25 × objective and 0.5 × Zoom for GFAP and pCREB images and 10 × objective and 0.6 × Zoom for Iba1 images and analyzed with ImageJ software. Quantitation of fluorescence was performed in the dorsomedial area around the striatal lesion and analyzed as percent area of antibody staining (GFAP and IBA1) or intensity (cumulative distribution and average intensity; pCREB).

Cells were washed with ice-cold PBS and lysed in a buffer containing 50 mM Tris (pH 7.4), 150 mm NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 1% Igepal, 1 mM polymethanesulphonyl fluoride (all from Sigma-Aldrich), and complete protease inhibitors (Roche Diagnostics, Mannhein, Germany); protein concentration was measured with a DC-protein assay (Bio-Rad, Hercules, CA, USA). Aliquots of total proteins were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis, blotted onto a PVDF membrane (Immobilon-P; Millipore), and blocked with 5% not-fat milk in 0.1 M Tris-buffered saline (pH = 7.4) for 30 min. Membranes were then incubated with the following primary antibodies: anti-pERK1/2 (1:2500; Santa Cruz Biotechnology), anti-pAkt (Thr308; 1:1000, Immunological Sciences), anti-TH (1:1500; Millipore), anti-pCREB (1:2000, Millipore), anti-c-Fos (1:2000, GeneTex), anti-p-p70S6K (1:1000; Cell Signaling), and anti-alpha-tubulin (1:300,000; Sigma-Aldrich). Blots were incubated with appropriate horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology) and signals were detected by enhanced chemiluminescence (ECL) (GeneSpin). The membranes were scanned and analyzed using gel-pro analyzer software (Media Cybernetics, Bethesda, MD, USA).

Samples were separated by SDS-PAGE, transferred to PVDF membrane, and blocked by 5% skimmed milk solution as previously described [40 (link),42 (link),76 (link)]. The following primary antibodies were used overnight in 1% skimmed milk solution: anti-UCP1 (1:750, R&D Systems, MAB6158), anti-pCREB (1:1000, Merck-Millipore, Burlington, MA, USA, 05-667), anti-CREB (1:1000, Abcam, Cambridge, UK, ab31387), anti-PGC1α (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA, H-300), anti-CKMT2 (1:1000, Novus Biologicals, Centennial, CO, USA, NBP2-13841), anti-CKB (1:1000, Novus Biologicals, NBP1-84460), anti-CRYAB (1:1000, Novus Biologicals, NB100-2519), anti-β actin (1:5000, Novus Biologicals, A2066), anti-OXPHOS (1:1000, Abcam, ab110411), anti-Aggrecan (1:1000, Novus Biologicals, NB100-74350), and anti-ID1 (1:1000, Novus Biologicals, JM92-13). HRP-conjugated goat anti-rabbit (1:10,000, Advansta, San Jose, CA, USA, R-05072-500) or anti-mouse (1:5000, Advansta, R-05071-500) IgG were used as secondary antibodies, respectively. Immunoreactive proteins were visualized by Immobilion Western chemiluminescence substrate (Merck-Millipore). Densitometry was carried out by FIJI.

Aβ1–42 (rPeptide, Aβ42, United States) 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 micro-infusion into prefrontal cortex. H89 (Sigma-Aldrich, United States) were dissolved in artificial cerebrospinal fluid (ACSF) for prefrontal cortex microinjection from day 8 to day 22 (once a day). A total of 60 mice were randomly divided into 5 groups (12 mice in each group) for behavioral tests. The groups were normal control + vehicle, Aβ1–42 + vehicle, Aβ1–42 + 4DmiRNA, Aβ1–42 + 4DmiRNA + H89, and Aβ1–42 + H89. After behavioral tests, mice were sacrificed for LTP recording (half brain) and an immunoblot assay (the other half brain).
The primary antibodies of anti-PSD95, anti-BDNF, and anti-SYN were purchased from Abcam Biotechnology Company (Abcam, Cambridge, MA). Anti-pCREB and anti-CREB were purchased from Merck Millipore (Millipore, Billerica, MA, United States). All the secondary antibodies (anti-rabbit lgG) were purchased from MultiSciences Biotech Co., Ltd. (MultiSciences, Hangzhou, China). The Cu-Zn/MnSOD and SOD ELISA kit was purchased from Shanghai Beyotime Biological Technology Co., Ltd. (Beyotime Sciences, United Kingdom).