Transfer unit

HEK293 lysates were obtained using RIPA lysis buffer (Sigma Aldrich) with phosphatase and protease inhibitor cocktails (Roche). The protein concentrations in the samples were determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific). A total of 20 µg of protein samples diluted in sample buffer containing 2-mercaptoethanol (Sigma-Aldrich), denatured for 10 min at 95 °C, were loaded in each well and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in a 10 or15% polyacrylamide gel cast using the TGX FastCast acrylamide kit (Bio-Rad). After electrophoretic separations, proteins were transferred to polyvinylidene fluoride (PVDF) membrane using a transfer unit (Bio-Rad) at 80 V for 1 h. After transfer, the PVDF membranes were blocked with 5% bovine serum albumin (BSA) for 2 h and incubated overnight at 4 °C with primary antibodies. These include: anti-pSer209eIF4E (Cell Signaling Technology #9741), anti- eIF4E (Cell Signaling Technology, #9742, anti-4E-BP1(Cell Signaling Technology #9452) and anti-β-actin (Cell Signaling Technology #8H10D10). Detection of the immunoreaction was performed with a Clarity western enhanced chemiluminescence substrate kit (Bio-Rad) using ChemiDoc MP imaging system (Bio-Rad).

Samples of the left hippocampus or cortex were homogenized in PBS containing complete protease inhibitors in a glass vesicle; the resulting homogenate was centrifuged at 12,000 rpm at 4°C for 20 min; and the protein concentrations of the supernatant thus obtained determined with the BCA protein assay kit. Proteins (30 μg) were subsequently separated by 8-10% SDS-PAGE and then blotted onto polyvinylidene difluoride (PVDF) membranes with a transfer unit (Bio-Rad Inc.). For the relative quantification of the proteins, these membranes were thereafter incubated with antibodies against α7 (1:1000), α4 (1:1500) and β2 (1:1000), cleaved caspase-3 (1:1000, Gene Tex, USA), Bcl-2 (1:1000, CST, USA), Bax (1:1000, CST, USA), Snap-25 (1:1000, Gene Tex, USA), Syn (1:1000, Gene Tex, USA) or β-actin antibody (1:5000, Gene Tex, USA) at 4°C overnight. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:5000, Gene Tex, USA) for 60 min. Finally, these membranes were incubated in ECL Plus (Pierce, Thermo Fisher Scientific) reagent and the signals thus obtained visualized by exposure to hyper-performance chemiluminescence film for 30 sec to 5 min.

Twelve serum samples from PB or BMA were collected for Western blotting. The protein content was quantitated using a protein assay kit (Pierce Biotechnology, Rockford, IL, USA), separated by 6% SDS-PAGE for catalase and 10% for glutathione peroxidase and β-actin, and transferred onto membranes using a transfer unit (Bio-Rad, Hercules, CA, USA). After blocking, the membranes were incubated with 1,000-fold diluted rabbit antibodies against catalase and glutathione peroxidase (Abcam, Cambridge, UK) or mouse antibodies against β-actin (Millipore, Temecula, CA, USA). After washing, the membranes were further incubated for 2 h with 10,000-fold goat anti-mouse IgG (Calbiochem, Millipore, Billerica, MA, USA) or goat anti-rabbit IgG (Millipore) conjugated to horseradish peroxidase. The membranes were then washed and rinsed with ECL detection reagents (Amersham Pharmacia Biotech, Amersham, Buckinghamshire, UK). The band images were photographed using ECL Hyperfilm (Amersham).

The levels of enzymes that cleave APP (BACE1, a β-secretase; ADAM10, a constitutive α-secretase; and BACE2, another β-secretase) and of synaptic proteins (SNAP25 and SYP) were determined by Western blotting. For this purpose, the brain tissues were homogenized in PBS containing complete protease inhibitors in a glass vesicle; the resulting homogenate was centrifuged at 12,000 rpm at 4 °C for 20 min; and the protein concentrations of the supernatant thus obtained determined with the BCA protein assay kit, and the protein sample of each group was 30 μg.
The proteins were subsequently separated by 10% SDS-PAGE and then blotted onto polyvinylidene difluoride (PVDF) membranes with a transfer unit (Bio-Rad Inc.). For the relative quantification of the proteins, these membranes were thereafter incubated with antibody against BACE1 (1:1000 dilution), ADAM10 (1:1500 dilution), BACE2 (1:500, dilution), SNAP-25 (1:1000 dilution), SYP (1:500 dilution), or β-actin antibody (1:5000, dilution), at 4 °C overnight. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:5000) for 60 min. Finally, these membranes were incubated in ECL Plus reagent, and the signals thus obtained visualized by exposure to hyper-performance chemiluminescence film for 30 s to 5 min.

Protein extract was applied to western blot analysis. For this purpose, the hippocampus was homogenized in lysis buffer containing complete protease inhibitor cocktail (1 M Tris-HCl (pH 8.0), 5 M NaCl, 10% Nonidet P-40, and 1 M 1,4-dithio-DL-threitol (DTT)) [27 (link)]. Lysate samples containing 30 μg of protein were fractionated by SDS-10% polyacrylamide gel electrophoresis and then blotted onto polyvinylidene fluoride membranes (Millipore, USA) with a transfer unit (Bio-Rad, USA). For the quantification of AChM1 receptor protein, these polyvinylidene fluoride membranes were thereafter incubated with primary antibodies as rabbit anti-rat-M1 polyclonal antibody at a 1 : 2000 dilution (Abcam, UK) and then incubated with a horseradish peroxidase-conjugated secondary antibody at a 1 : 5000 dilution. The membranes were put into chemiluminescence (ECL) reagent and exposed to film. Band densities were determined with image densitometer software. GAPDH was used as a housekeeping protein to normalize the protein load.