Gs 800 system

Protein solutions were extracted from hippocampal tissues; then, equal amounts of protein were attached onto a sodium dodecyl sulfate-polyacrylamide gel to be separated by electrophoresis according to their molecular weight. After electrophoresis, proteins were transferred to a nitrocellulose membrane (Amersham Bioscience, Piscataway, NJ, USA) using semidry transfer apparatus (Bio-Rad, Hercules, CA, USA). The membranes' nonspecific binding sites were then blocked by soaking in 5% skimmed milk. Next, the membranes were incubated at 4°C overnight on a roller shaker with solutions containing antiphosphorylated tau (1 : 10000, Cat. # ab109390), anti-GSK-3β (1 : 5000, Cat. # ab32391), and anti-mTOR (1 : 10000, Cat. # ab134903), which were obtained from Abcam (Cambridge, MA, USA). The membranes were then washed and incubated with the horseradish peroxidase-conjugated secondary antibody solution. Finally, the blots were developed with enhanced chemiluminescence detection reagents (Amersham Biosciences, Arlington Heights, IL, USA). Scanning laser densitometry (GS-800 system, Bio-Rad, Hercules, CA, USA) was used to determine the quantities of the target proteins. The results were normalized with β-actin protein expression and expressed as arbitrary units.

Following striatal protein quantification (Bio-Rad Protein Assay Kit, CA, USA), 10 μg proteins of each sample were separated by SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane using a semi-dry transfer apparatus (Bio-Rad, CA, USA). Membranes were then soaked in 5% non-fat dry milk to block non-specific binding sites. Afterward, the membrane was incubated with anti- p-S133 CREB (1:250; cat#: PA1-851B), anti- pS473 Akt (1:100; cat#: OMA1-03061) and anti-TLR4 (1:100; cat#: MA5-16216) polyclonal antibody (ThermoFisher Scientific, MA, USA) overnight at 4°C on a roller shaker. Next, membranes were probed with horseradish peroxidase-conjugated goat anti-rat immunoglobulin (Dianova, Hamburg, Germany). Finally, the blots were developed with enhanced chemiluminescence detection reagent (Amersham Biosciences, IL, USA). Protein was quantified by densitometric analysis using a scanning laser densitometer (GS-800 system, Bio-Rad, CA, USA). Results were expressed as arbitrary units (AU) after normalization for β-actin protein expression.