Digital science 1d software

The protein concentrations of the brain extracts and cell lysates were determined by BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Then the samples were mixed with sample buffer containing 50 mM Tris-HCl (pH 7.6), 2% SDS, 10% glycerol, 10 mM dithiothreitol, and 0.2% bromophenol blue and boiled for 5 min. Boiled protein samples (15–20 μg per lane) were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membranes. The membranes were detected by using anti-rabbit or anti-mouse IgG conjugated to IRDye (800CW; Li-cor Biosciences, Lincoln, NE, USA) for 1 h at room temperature and visualized using the Odyssey Infrared Imaging System (Li-cor Biosciences, Lincoln, NE, USA). The protein bands were quantitatively analyzed by Kodak Digital Science 1D software (Eastman Kodak Company, New Haven, CT, USA).

The animals were decapitated after the spatial memory retention test. The hippocampi were rapidly removed and homogenized at 4 °C using a Teflon glass homogenizer in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM NaF, 1 mM Na₃VO₄, 5 mM EDTA, 2 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride. The extract was mixed with sample buffer (3:1, v/v) containing 200 mM Tris-HCl, pH 7.6, 8% SDS, 40% glycerol, 40 mM DTT, boiled for 10 min, and then centrifuged at 12,000 × g for 10 min at 25 °C. The supernatant was stored at −80 °C for western blotting analysis. The protein concentration in the supernatant was estimated using a BCA kit according to the manufacturer’s instructions. The proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk dissolved in TBS-Tween-20 (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.2% Tween-20) for 1 h and probed with primary antibody at 4 °C for overnight. Then, the blots were incubated with anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase (1:5000) for 1 h at 37 °C and visualized with enhanced chemiluminescence. The blots were quantitatively analyzed using the Kodak Digital Science 1D software (Eastman Kodak Co., New Haven, CT, USA).

Hippocampal tissues were mixed and homogenized with sample buffer (pH 7.6; 50 mM Tris-HCl, 10 mM dithiothreitol, 2% sodium dodecyl sulfate, 10% glycerol, and 0.2% bromophenol blue) on ice and boiled for 10 minutes. Thirty micrograms of protein was heated at 95°C for 5 minutes and then cooled on ice for 5 minutes. The protein was fractionated on a 4–12% SDS-PAGE Bis-Tris gel using an SDS-PAGE system (Bio-Rad, CA, USA). The proteins were transferred onto nitrocellulose membranes (Whatman, Kent, UK). Following blocking in 5% nonfat milk in 0.01% Tween PBS (PBST), the membranes were incubated with optimally diluted primary antibodies overnight at 4°C. Following washing with PBST, the membranes were incubated with the appropriate anti-mouse or anti-rabbit secondary antibody diluted 1:10,000 for 1 h at room temperature and visualized using the Odyssey Infrared Imaging System. The protein bands were quantitatively analyzed by Kodak Digital Science 1D software (Eastman Kodak Company, New Haven, CT, USA).

For western blotting, samples were boiled at 100 °C for 5 min in the loading buffer (50 mM Tris-HCl, pH 7.6, 2% SDS, 10% glycerol, 10 mM DTT and 0.2% bromophenol blue). The proteins were electrophoresed in 10% SDS-PAGE and the separated proteins transferred to nitrocellulose membranes (Amersham Biosciences, Pittsburgh, USA). The membranes were then blocked with 5% non-fat milk dissolved in TBS-Tween-20 (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.2% Tween-20) for 1 h and probed with primary antibody at 4 °C overnight. Then the blots were detected using anti-rabbit or anti-mouse IgG conjugated to IRDye (800CW; Licor Biosciences, Lincoln, NE, USA) for 1 h at room temperature and visualized using the Odyssey Infrared Imaging System (Licor Biosciences). The protein bands were quantitatively analyzed by Kodak Digital Science 1D software (Eastman Kodak Company, New Haven, CT, USA).
To analyze protein–protein interactions, co-immunoprecipitation experiments were performed using wild-type or tau Ko mice brain homogenates. Specified antibody and protein G agarose were incubated with the homogenates overnight at 4 °C. The resins were washed for three times with PBS. After elution by 2 × loading buffer, and boiled at 95 °C for 5 min, the bound proteins were analyzed by western blotting.

The nuclear and cytoplasmic protein extraction kit (Beyotime, Shanghai, China) was used to extract the total protein from the treated SH‐SY5Y neuronal cells, and then quantified with a BCA kit (Beyotime, Shanghai, China). Approximately 40 μg of protein was loaded and separated with a 12% SDS‐polyacrylamide gel (SDS‐PAGE) and then further transferred to the PVDF membrane (Millipore, MIT, USA). After blocking the non‐specific proteins using 5% non‐fat milk, the membrane was incubated with primary antibodies against p53 (1:2000, #2524, CST, Boston, USA), p21 (1:3000, #2947, CST, Boston, USA), Nrf2 (1:1500, #12721, CST, Boston, USA), and β‐actin (1:10000, #4970, CST, Boston, USA) at 4 °C overnight, followed by being incubated with anti‐mouse IgG, HRP‐linked antibody (1:2000, #7076, CST, Boston, USA), and anti‐rabbit IgG, HRP‐linked antibody (1:3000, #7074, CST, Boston, USA) at room temperature for 2 h. Lastly, the blots were incubated with the ECL reagents (Beyotime, Shanghai, China) and exposed to Tanon 5200‐multi (Tanon, Shanghai, China). The bands of the blot were scanned, selected, and the sum optical density was quantified using the Kodak Digital Science 1D software (Eastman Kodak Company, USA). Data were exported for statistical analysis and normalized to β‐actin.