Ab134101

The brain tissues of 3 mice from each group were collected and fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4 μm. The oven was heated to 56 °C, and the selected sections were baked for 1 h. The brain sections were deparaffinized in xylene and rehydrated. For staining, differentiation, and antiblue staining with hematoxylin, the sections were immersed in hematoxylin for 3 min, and then differentiated with 1% hydrochloric acid for 10 s. After eosin had been impregnated for 3 min, the sections were then dehydrated by gradient. Finally, the film was sealed and photographed under a microscope. The immunohistochemical assay procedure was the same as that of our previous experiment [9 (link), 12 ]. The brain slices was incubated with the following primary antibodies: Aβ₄₂ (1:1500, ab201060, Abcam, USA), InR (1: 500, ab5500, Abcam, USA), IRS2 (1:500, ab134101, Abcam, USA), GSK3β (1:500, ab32391, Abcam, USA), p-GSK3β (1:500, ab75814, Abcam, USA). The slices were observed under a × 20 objective lens of an optical microscope. The Aβ plaques was observed under a × 5 objective lens of an optical microscope.

The total protein was extracted from the liver, soleus muscle and ovary tissues of each group of mice, and the concentration of the sample protein was determined using the BCA kit (Beyotime, Shanghai, China). Then, 40 μg protein with 10 μL protein loading buffer was transferred to a 5 × sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). When the bromophenol blue dye solution completely ran into the separation gel, the voltage was increased to 180 V until the bromophenol blue dye solution approached the lower edge of the gel. Then, the electrophoresis was stopped, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was sealed with 5% skim milk powder for 1.5 h then incubated with corresponding primary antibodies (anti-β-actin, ab8226, 1:5000; INSR, ab283689, 1:1000; IRS-1, ab46800, 1:1000; p-IRS-1 (phospho Tyr632), ab109543, 1:1000; IRS-2, ab134101, 1:1000; and p-IRS-2 (phospho ser1149), ab178103, 1:1000, Abcam, Cambridge, UK) overnight at 4°C by shaking. The next day, the membrane was washed, then incubated with secondary antibody. Later, enhanced chemiluminescence (ECL) luminescent solution was applied to visualize the protein band in a dark room. Finally, Image J 180 software was employed to measure the grey value of each band of WB.

Protein lysates were prepared by freezing hippocampal tissues or cell homogenates with RIPA lysates containing proteases and phosphatase inhibi tors. After centrifuged at 12,000 rpm for 15 min at 4 °C, the supernatant was collected. Protein concentrations were measured by BCA kit (Bioss, China). The proteins were loaded and separated by SDS-PAGE and then transferred to PVDF membrane. The membrane was blocked by 5% defatted milk for 1 h. The membrane was then incubated with the following primary antibodies: Aβ₄₂ (1:1500, ab201060, Abcam, USA), InR (1:1000, ab5500, Abcam, USA), p-InR (1:1000, ab60946, Abcam, USA), IRS2 (1:1000, ab134101, Abcam, USA), p-IRS2 (1:1000, ab3690, Abcam,USA),PI3K (1:1000, ab151549, Abcam, USA), Akt (1:1000, ab179463, Abcam, USA), p-Akt (1:1000, ab8805, Abcam, USA), GSK3β (1:1000, ab32391, Abcam, USA), p-GSK3β (1:1000, ab75814, Abcam, USA), GLUT1 (1:1000, ab652, Abcam, USA), GLUT3 (1:1000, ab41525, Abcam, USA),and β-actin (1:10,000, AY0573, Abways, China) at 4 °C overnight. After rinsing, the membrane was probed with secondary antibody at room temperature for 1 h. Finally, enhanced chemiluminescence reagent detection system was used to visualize the protein expression. Protein blots were quantified using ImageJ software and results were expressed quantitatively after normalizing blots with β-actin.

Cellular proteins were extracted and equal amounts of protein fractionated by on 10% SDS-PAGE. The proteins were electrotransferred onto a PVDF membrane and blocked with 5% non-fat dry milk in PBST for 1 h. Antibodies specific for PDK-1 (ab110025, mouse monoclonal, 1:1000, Abcam, Cambridge, MA, U.S.A.), YAP (sc-376830, mouse monoclonal, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), insulin receptor substrate 2 (IRS2) (ab134101, rabbit monoclonal, 1:1000, Abcam), and β-actin (ab6276, mouse monoclonal, 1:10000, Abcam) were used for immunoblot assays. The proteins were developed by an enhanced chemiluminescence (ECL) kit.