Tbst solution

The hippocampus sample was obtained as in Section 4.5.2. RIPA buffer was added for tissue lysis and extraction of total protein. Protein concentration determination was performed using a BCA kit, and the samples were separated by electrophoresis on a 5–10% polyacrylamide gel. After separation, the proteins were transferred to a 0.45 μm PVDF membrane by the wet transfer method using constant piezoelectricity at 100 V for 1.5 h. The membrane was blocked in 5% skimmed milk for 2 h, incubated overnight with the primary antibody, washed in a TBST solution (SolarBio, Beijing, China) for 3 by 10 min, incubated in the secondary antibody at room temperature for 1 h on the shaker, washed a further three times in TBST solution, and placed on plastic wrap. Developer from the ECL chemiluminescence detection kit was added evenly, and the membrane was allowed to stand. The filter was blotted dry for 1 min, and an Azure multifunctional molecular imaging system (C600, Azure, USA) was used for detection of the labelled proteins. GAPDH was used as the control for protein loading and transfer efficiency Gray value analysis was performed using Image J software (The National Institutes of Health, Bethesda, MD, USA).

Nasal mucosa tissues were cut and centrifuged to extract proteins using TRIzol reagent (Invitrogen, Carlsbad, California, U.S.A.). BCA kit (Thermo Scientific, Waltham, Massachusetts, U.S.A.) was used to measure protein concentration, and protein ladder (Invitrogen, Carlsbad, California, U.S.A.) was separated from protein by SDS/PAGE. Next, the proteins were transferred to PVDF membranes (Sigma–Aldrich, St. Louis, Missouri, U.S.A.), which were blocked by 5% albumin. The protein membranes were incubated first with primary antibody (Table 1) was used to incubate at 4°C for 12 h and then with secondary antibody (ab7090, Abcam, Cambridge, Massachusetts, U.S.A.) at 25°C for 3 h. Primary antibody and secondary antibody were dissolved in TBST solution (Solarbio, Beijing, China) following the instructions. ECL kit (Sigma–Aldrich, St. Louis, Missouri, U.S.A.) was used to stain the membranes in the dark. The protein flaser were taken by fluorescence imaging system (CliNX, Shanghai, China).

48 h after cells were transfected in various groups, they were rinsed with cold PBS (Thermo Fisher, USA) in triplicate and lysed on ice for 10 min with whole protein lysate. Protein quantification was done with BCA quantitative kit (Thermo Fisher, USA). 10 μL loading buffer was supplemented, and proteins were boiled for 10 min at 95°C. SDS-PAGE was performed at 100 V. After the electrophoresis, proteins were transferred to the NC membrane at 100 mA and 120 min, sealed with 5% BSA/TBST for 60 min, followed by incubation with primary antibodies at 4°C overnight. After incubation, the membrane was washed in a shaking table with 1 × TBST solution (Solarbio, Beijing, China) 5 min × 3 times at room temperature. Goat anti-rabbit IgG labelled with horseradish peroxidase was used for hybridization for 120 min at room temperature. Membrane was washed with TBST for 20 min × 3 times and. Then, luminescence reaction was performed with ECL kit (Solarbio, Beijing, China). Protein imprinting was observed by taking photos. The assay was done in three replicates. Antibody information was exhibited in Table S3.