Anti phospho creb

The γ-secretase inhibitors [N-[N-(3,5-Difluorophenacetyl-Lalanyl)]-S-phenylglycine t-butyl ester (DAPT)], L-685,486, [1,2-bis(o-Aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM)] and SB202190 were purchased from Calbiochem (La Jolla, CA). 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT), Cadmium chloride (CdCl₂), N-acetylcysteine (NAC) and N-[2-(Cyclohexyloxy)-4-nitrophenyl] methanesulfonamide (NS-398), Bicinchoninic acid protein assay kit were purchased from Sigma (Saint Louis, MO). Anti-cleaved parp (#9545, Asp214/215), anti-cleaved caspase-3 (#9664, Asp175), anti-phospho ERK1/2 (#9101, Thr202/Tyr204), anti-ERK1/2 (#9102), anti-stress-activated protein kinase (SAPK)/JNK (#9252), anti-phospho SAPK/JNK (#9251, Thr183/Tyr185), anti-p38 MAPK (#9212) and anti-phospho p38 MAPK (#9211, Thr180/Tyr182), anti-CREB (#9197), anti-phospho CREB (#9198, Ser133), anti-AKT (#9272) and anti-phospho AKT (#9275, Thr308) antibodies were from Cell Signaling Technology (Beverly, MA). Anti-COX-2 (sc-1745) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-GFP antibody (A11122) obtained from Molecular Probes (Eugene, OR). Anti-activated notch-1 antibody (ab8925) was from Abcam (Cambridge, MA).

Antigen retrieval and IHC analyses were performed as described previously [32 (link)]. We used the following antibodies: anti-14-3-3ζ (Santa Cruz); anti-LDHA, anti-CREB, anti-phospho-CREB, anti-ERK, anti-phospho-ERK, anti-cleaved caspase 3 (Cell Signaling); and anti-MCM (Epitomics). For TMA, we used a 70-case, 208-core breast cancer tissue microarray (catalog no. BR208, US Biomax Inc.).

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.

Hippocampus samples obtained at the end of the animal experiment were homogenized in lysis buffer containing 1% protease inhibitors. Approximately 15 µg proteins were moved to a new tube and incubated at 100 °C for 5 min for denaturation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was run at 120 V for 45 min. The proteins were transferred to polyvinylidene difluoride membranes and then blocked with 5% non-fat dry milk buffer for 60 min at room temperature (RT). Blots were incubated with anti-phospho-p44/42 mitogen-activated protein kinase (MAPK), anti-p44/42 MAPK (ERK1/2), anti-CREB, anti-phospho-CREB, anti-AKT (Cell Signaling Technology, Boston, MA, USA), and anti-phospho-AKT (Abcam, Hong Kong, China) antibodies overnight at 4 °C. After washing three times, the membranes were incubated with secondary antibody for 45 min at RT. ^eECL Western Blot Kit (Beijing CoWin Biotechnology, Beijing, China) was used to develop the bands.

Aβ42 peptide was purchased from American Peptide Company (Sunnyvale, CA, USA) and was prepared as previously described (Byun et al., 2015). It was dissolved in hexafluoroisopropanol for 72 h at room temperature (RT) and lyophilized. The peptide was then dissolved again in dimethylsulfoxide. Anti‐ZO‐1, anti‐Claudin 5 (Thermo Fisher Scientific, Waltham, MA, USA), anti‐GAPDH (Abcam, Cambridge, MA, USA), anti‐GFP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti‐CD31 (R&D Systems, Minneapolis, MN, USA), antiphospho CREB (Cell signaling Technology, Danvers, MA, USA), anti‐CREB (Cell signaling Technology), and anti‐Annexin A1 (Invitrogen, Carsbad, CA, USA) were used for Western blot analysis, and Anti‐ZO‐1 (Thermo Fisher Scientific) was used for immunofluorescence images. Y27632, MK801, and l‐glutamate were purchased from Sigma‐Aldrich Co. (St. Louis, MO, USA), Rhosin was purchased from Merck Millipore (Billerica, MA, USA), and human Annexin A1 (ANXA1) recombinant protein was purchased from MyBioSource (San Diego, CA, USA). For experiments, bEnd.3 cells and/or pericytes were treated with Aβ42 (2 μm and/or 5 μm; from 0 to 24 h, differently for each experiment), ANXA1 (1 μg mL⁻¹ and/or 2 μg mL⁻¹; for 24.5 h), Y27632 (30 μm; for 24 h), Rhosin (10 μm; for 24 h), l‐glutamate (30 μm; for 30 min or 24 h), and MK801 (10 μm; for 30 min or 24 h).