Anti creb

Antibodies anti-HA (Babco), anti-RSK2, anti-APPL1, anti-Rab7, anti-TrkB (Santa Cruz Bio-technology), anti-PLD1, anti-ERK, anti-phospho-ERK, (New England BioLabs), anti-β-tubulin, anti-CREB (Millipore), anti-GAPDH, anti-phospho-CREB (Ser-133), anti-mTOR, anti-phospho-mTOR (Ser-2481), anti-phospho-S6K (Thr-389), anti-phospho-S6K (Thr-421/Ser-424), anti-PEA15 (Cell Signalling), anti-Rab5 (Transduction Laboratories) were used. Plasmids have been described previously15 (link)17 (link). ON-TARGETplus siRNA were obtained from Darmacon and BDNF was from Invitrogen.

Nuclear extracts of mouse PF cortex were prepared in 0.25 M sucrose buffer (sucrose 0.25 M, Tris 50 mM, EDTA 1 mM, imidazole 3 mM, pH 7.0 + proteases inhibitor cocktail). Samples were centrifuged 10 min at 250 g and nuclear fraction was resuspended in Laemmli buffer. All samples were sonicated before protein assay (BCA Pierce, Thermoscientific) and Western blotting was performed on 20 µg of protein lysates. Membranes were incubated overnight at 4°C with the primary antibodies; anti-HDAC2 1∶1500 (Abcam), anti-H3 1∶10000 (Millipore), anti-tubulin 1∶4000 (Sigma), anti-CREB and phospho-CREB 1∶1000 (Millipore). Washes with PBS-Tween (0.005%) were followed by incubation with secondary antibody (1∶10 000 anti-mouse or anti-rabbit IgG) (GE Healthcare) coupled to horseradish peroxidase and revealed by ECL. For quantification, the membranes were stripped and reincubated with an anti-tubulin or an anti-H3 antibody. Immunoreactive bands were quantified with an electrophoresis Gel Doc 2000 imaging system coupled to a Quantity one software (Bio-Rad).

Tax was detected using sera from HTLV-1 infected individuals (kindly provided by Dr Gessain, Institut Pasteur, Paris, France) or the anti-Tax monoclonal antibody (mab) 168-A51 (NIH AIDS Research and Reference Reagent Program, USA). The following primary antibodies were used: anti-GFP recognizing GFP as well as the YFP and YPET variants (Roche Applied Science), anti-OGT (Sigma, DM-17 06264), anti-OGA (Santa Cruz, sc135093 or Sigma, SAB4200311), anti-O-GlcNAc (Abcam, RL2), anti-CREB (Millipore, CS 203204), anti-phospho CREB ser133 (Millipore, CS 204400), anti-actin (Santa Cruz, sc1616), anti-tubulin (GeneTex, GT114) and GAPDH (Santa Cruz, sc32233). HRP-conjugated anti-human, anti-mouse and anti-rabbit IgG (Promega) were used as secondary antibodies. Thiamet G (Sigma, SML 0244) was used at 10μM concentration.

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).

The following commercially available antibodies were used for chromatin immunoprecipitation experiments: Anti-H3K4me3 (Millipore, catalog number 04–745; RRID:AB_1163444), Anti-H3K36me3 (Abcam, catalog number ab9050; RRID:AB_306966), Anti-CREB (Millipore, catalog number 06–519; RRID:AB_310153), Anti-PolII (Ser5-phosphorylated) (Abcam, catalog number ab5131; RRID:AB_449369), Anti-PolII (Ser2-phosphorylated) (Abcam, catalog number ab5095; RRID:AB_304749), Anti-CBP/p300 (Millipore, catalog number 05–257; RRID:AB_11213111), Anti-C/EBPß (Santa Cruz, catalog number sc-150; AB_2260363) or Anti-ATF4 (Santa Cruz, catalog number sc-200; RRID:AB_2058752). The following commercially available antibodies were used for microscopy experiments: anti-RFP (Invitrogen R10367; RRID:AB_2315269), anti-GFP (Aves Labs GFP1010; RRID:AB_2307313), goat anti-rabbit Alexa 555 (Invitrogen A21428; RRID: AB_10561552) and goat anti-rabbit Alexa 488 (Invitrogen A11039; RRID:AB_142924). The following cell lines were used: rat H19-7 (ATCC CRL-2526; RRID: CVCL_H781), mouse Neuro-2a (ATCC CCL-131; RRID:CVCL_0470), human HEK-293FT (Invitrogen R70007; RRID:CVCL_6911), human HEK-293T (ATCC CRL-3216; RRID:CVCL_0063).