Anti psd95

Aliquots of protein lysates (30 µg) prepared from hemibrains homogenized in RIPA buffer were loaded in Criterion XT 4–20% Bis-Tris gels and transferred onto PVDF membrane (0.45μm; Millipore, Billerica, MA, USA). Membranes were probed with anti-6E10 (1:2000, cat#9320–500, Covance, Princeton, NJ, USA), anti-Synapsin-1 (1:1000, cat#106103, Synaptic Systems, Göttingen, Germany), and anti-PSD-95 (1:1000, cat#MAB1596, Millipore, Billerica, MA, USA) antibodies and subsequently incubated with anti-rabbit or anti-mouse HRP-conjugated secondary antibodies (1:2000, cat#PI-1000 or PI-2000, Vector laboratories). Normalization was achieved using GAPDH antibody (1:5000, cat#sc32233, Cruz Biotechnology, Dallas, TX, USA). Membranes were developed with ECL Western blotting substrate (Pierce, Rockford, IL, USA) using the Fujifilm LAS-3000 developer (Stamford, CT, USA). Integrated density of immunoreactive bands was measured using Fiji software (ImageJ).

Antibodies, plasmids and other reagents were obtained from the following resources. Antibodies: anti-GluA1Ct (rabbit) was made by OriGene; anti-GluA2/3 (rabbit), anti-NR1Ct (mouse), anti-PSD95 (mouse), anti-eIF4e (rabbit) were from Millipore; anti-αTubulin (mouse) was from Sigma-Aldrich; anti-phosphoAMPKαThr172 (rabbit), anti-Phospho-AktSer473 (rabbit), anti-eIF4g (rabbit) and anti-Akt (rabbit) were from Cell Signaling; anti-SIRT1 (rabbit) was from Abcam. Chemicals: Resveratrol, MG132, Leupeptin, Cycloheximide, Anisomycin, Actinomycin D, Compound C, LY 294002, STO609, Dimethyl sulfoxide (DMSO) were from Sigma-Aldrich; EX527 and 4EGI-1 were from Millipore; 5-aminoimidazole-4-carboxamide-1-β-D-riboside (AICAR) was from Enzo Life Sciences; Plasmids and siRNAs: pcDNA was purchased from Invitrogen; EGFP was purchased from Clontech Laboratories. αAMPK kinase dead (AMPK K.D.) and PI3 kinase dominant negative form (PI3K D.N.) were generously provided by Prof. Lewis C. Cantley (Harvard Medical School, Boston, MA); G-CaMP3 was generously provided by Prof. Loren L. Looger (Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA) via his deposit at Addgene (#22692); siRNAs (Scramble and SIRT1) were purchased from Santa Cruz Biotechnology.

Equal amounts of protein (30 μg) were separated by electrophoresis in precast 4-12% Bis-Tris Gels (Bio-Rad) and transferred to activated/pre-wetted PVDF membranes. The membranes were hybridized with the following primary antibodies as indicated: AT8 anti-P-tau pSer202/Thr205 (1:500, Thermo Scientific, #MN1020); anti-P-tau pS396 (1:1000, Abcam, #ab109390); anti-total tau T46 (1:1000, Thermo Scientific, #13-6400); anti-GAPDH (1:2000, Santa Cruz, #sc-32233); anti=P-CaMKII (1:1000, Abcam, #ab32678); anti-PSD-95 (1:1000, Millipore, #7E3-1B8), C1q (1:1000, Abcam, #ab182451). Secondary antibodies included: peroxidase labeled anti-mouse IgG (1:2000, Vector Laboratories); peroxidase labeled anti-rabbit IgG (1:2000, Vector Laboratories); peroxidase labeled anti-rat IgG (1/2000, Vector Laboratories). ECL (Pierce®) was used to reveal the immunoreactive proteins, and images were acquired using a Fujifilm ImageReader LAS-4000. Membranes were stripped using a stripping buffer (Thermo Scientific) when required. Luminescent immunoreactive protein bands were quantified using Fiji software (ImageJ).

Recombinant human BDNF, leupeptin, aprotinin, phenyl-methylsulfonyl fluoride, pepstatin A, soybean trypsin inhibitor, sodium fluoride, sodium vanadate, glycerophosphate, 2-mercaptoethanol, NMDA, glycine, Tween 20, NP-40, and Histopaque-1077 were from Sigma. Anti-PSD-95 (05494) was from Millipore. Anti-TrkB (SC-8316), -pTrkB (SC8058), -BDNF/pro-BDNF (SC-2098), -NT3 (SC-547), -NT4 (SC-545), -phosphotyrosine (SC-508), -Erk2 (SC-154, SC-81457), -pErk2 (SC-7383), -pAkt1 (SC-7985-R), -Akt1 (SC-65487), -PLC (SC7290), -NR1 (SC-9058), -NR2A (SC-9056), -actin (SC-376421), -β-actin (SC-47778), and -Arc (SC365736) were from Santa Cruz Biotechnology. Seize-X immunoprecipitation kit, antigen elution buffer, Bind NeutrAvidin, high binding capacity coated 96-well plates, and West Pico chemiluminescent reagents were from Pierce-Endogen. Bradford reagent, SDS-PAGE reagents, and prestained molecular weight markers were from ThermoFisher. Protease inhibitors (EDTA-free) and protein phosphatase inhibitor tablets were from Roche. BDNF was reconstituted according to the manufacturer’s instruction. To avoid freezing damage, 10% glycerol was added to achieve 10 ng/L BDNF and it was stored at −80°C until use. All other test agents were made fresh according to the manufacturer’s recommendation. The DMSO concentration in the incubation medium was 1% when used.