2.13 Western blot
Cortexes or cells were lysed with RIPA buffer supplemented with protease
inhibitor, NaF, PMSF and phosphatase inhibitor. The protein
concentrations were determined using a BCA kit according to the
manufacturer’s instructions. Proteins were separated by SDS/PAGE and
then transferred to PVDF membranes to incubated with rabbit anti-Lf
(Millipore, 07-685, 1:1000; recognizes mouse and human Lf), rabbit
anti-human Lf (Abmart, T59526, 1: 1000), rabbit anti-APP695 (Cell
Signaling Technology, 2452, 1: 1000), rabbit anti-C-APP (Sigma,
SAB4200535, 1:1000), rabbit anti-BACE1 (Abcam, ab183612, 1: 1000),
rabbit anti-AMAD 10 (Cell Signaling Technology, 14194, 1: 1000), rabbit
anti-presenilin-1 (PS1; Cell Signaling Technology, 5643, 1: 1000), mouse
anti-soluble amyloid precursor α (sAPPα; Immuno-Biological Laboratories,
11088, 1: 500), mouse anti-soluble amyloid precursor β (sAPPβ;
Immuno-Biological Laboratories, 10321, 1: 500), mouse
anti-insulin-degrading enzyme (IDE; Santa Cruz, sc-514458, 1:500), goat
anti-apolipoprotein E (APOE; Santa Cruz, sc-6384, 1: 500), rabbit
anti-low-density lipoprotein receptor-related protein 1 (LRP1; Abcam,
ab92544, 1: 8000), rabbit anti-advanced glycation end products (RAGE;
Sigma, SAB2105049, 1: 1000), rabbit anti-Oligomer (Millipore, AB9234, 1:
1000), mouse anti-tau (Sigma, 577801, 1:5000), rabbit anti-p-tau
(Thr181) (Cell Signaling Technology, 62672, 1: 2000), rabbit anti-p-tau
(Ser202) (Cell Signaling Technology, 39357, 1: 2000), rabbit anti-p-tau
(Thr231) (Cell Signaling Technology, 71429, 1: 2000), rabbit anti-p-tau
(Ser396) (sigma, SAB4504557, 1: 2000), rabbit anti-p-tau (Ser404) (Cell
Signaling Technology, 35834, 1: 2000), rabbit anti-glycogen synthase
kinase 3α/β (GSK3α/β; Cell Signaling Technology, 5676, 1: 3000), rabbit
anti-p-GSK3α/β (Ser21/9) (Cell Signaling Technology, 8566, 1: 4000),
rabbit anti-p38 (Cell Signaling Technology, 8690, 1: 2000), rabbit
anti-p-p38 (Cell Signaling Technology, 4511, 1: 2000), rabbit
anti-cyclin-dependent kinase 5 (CDK5; Cell Signaling Technology, 14145,
1: 1000), rabbit anti-p-CDK5 (Cell Signaling Technology, 19051, 1:
1000), rabbit anti-PP2Ac (Cell Signaling Technology, 2038, 1: 1000),
rabbit anti-p-Erk1/2 (Cell Signaling Technology, 4370, 1: 1000), rabbit
anti-Erk1/2 (Cell Signaling Technology, 4695, 1: 1000) and mouse
anti-β-actin (Sigma, A2228, 1: 10000) overnight at 4 °C. Membranes were
washed with TBST and subsequently incubated with horseradish peroxidase
(HRP)-labeled secondary antibodies for 1 h at room temperature. Enhanced
chemiluminescence (ECL) kits (Tanon, 180-5001) and Chem Doc XRS with
Quantity One software (Bio-Rad, 5500) were applied to detect blots. Data
from the bands were determined using Image J software.