Ab32216

Proteins were sequentially extracted from brain tissues with PBS, radioimmunoprecipitation assay (RIPA), and 5 M guanidine buffer in the presence of protease and phosphatase inhibitors as we described previously (68 (link)). The RIPA fractions were used for the Western blot analysis. Equal amounts of proteins (20 μg) were loaded onto 4 to 20% TGX gels (Bio-Rad), separated by gel electrophoresis, and transferred onto polyvinylidene difluoride membranes. Blots were probed with the APP (1:1000; Invitrogen, 51-2700), BACE-1 (1:1000; Cell Signaling Technology, 5606), anti-Aβ 82E1 (1:1000; IBL-AMERICA, IBL10323), IDE (1:1000; Abcam, ab32216), NEP (1:1000; Abcam, 208778), Iba1 (1:1000; Abcam, ab178846), and β-actin (1:20,000; Sigma-Aldrich, A1978) antibodies. Signals were visualized by chemiluminescence [ECL Select (GE Healthcare) or TMA-6 ECL detection kit (Lumigen)]. Blots were quantified by densitometry with ImageJ software [National Institutes of Health (NIH)]. Results were normalized by β-actin levels and given as a fold change relative to Abi3^+/+ genotype.

Liver samples were collected and homogenized. After centrifugation at 12000 rpm for 30 min, 4°C, the protein was determined using Bradford reagent. For SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis, all samples were treated with a Laemmli buffer containing dithiothreitol. After heating to 100°C for 5 min, proteins were separated by electrophoresis in a 10% polyacrylamide gel. The transfer to nitrocellulose membranes was performed in a Trans Blot transfer for 2 h in 100 V, with a tris/glycine buffer. After, the membranes were blocked with 5% BSA for 1 h and were then incubated with polyclonal antibodies against IDE (Abcam, cat. Ab32216). Tubulin (Sigma Aldrich, cat. 6074) was used as a control. Visualization of specific protein bands was performed by incubating the membranes with appropriate secondary antibodies. Protein bands were visualized using the Amersham Imager 600 (GE Healthcare Life Sciences, Buckinghamshire, United Kingdom), which detected chemiluminescence. The band intensities were quantified with ImageJ software (National Institutes of Health, Bethesda, MD, United States). Full scans of the entire original nitrocellulose membranes are available on Supplementary information (Supplementary Figures 1–4).

Cerebral cortex extracts were loaded onto 10–16% Tris/tricine SDS gels and transferred to nitrocellulose membranes before overnight incubation with one of the following primary antibodies: mouse anti-β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) (1:1000; Millipore, #MAB5308), anti-PS1 (1:1000; Sigma-Aldrich, #PRS4203), anti-neprilysin (NEP) (1:1000; Millipore, #AB4348), anti-insulin-degrading enzyme (IDE) (1:1000; Abcam, #ab32216), anti-low-density lipoprotein receptor-related protein (LRP1) (1:1000; Abcam, #ab92544), anti-APP (C-terminal) (1:1000; Sigma-Aldrich, #A8717), mouse anti-total Aβ (Aβ_1–16) (1:1000; Covance, #803001), and anti-apoE (1:1000; Abcam, #ab20874) or GAPDH (1:1000; Bioworld; #AP0063). Horseradish peroxidase-conjugated secondary antibodies (Vector Laboratories) were used, and bands were visualized using ECL plus detection system. GAPDH was utilized as an internal control for protein loading and transfer efficiency.

The cells growing on slides were fixed with 4% of paraformaldehyde, washed three times with PBS, and then permeated with 0.5% Triton X-100 (diluted with PBS) for 20 min at room temperature. After washing the slides three times with PBS, 1% bovine serum albumin (BSA) as a blocking agent was cultured with cells at room temperature for 30 min. Then, absorbing the sealed solution with absorbent paper, a sufficient amount of anti-CD68 (M1 marker, ab237968, Abcam), anti-iNOS (M1 marker, ab49999, Abcam), anti-CD206 (M2 marker, ab64693, Abcam), anti-arginase1 (M2 marker, ab91279, Abcam), and anti-insulin degrading enzyme (ab32216, Abcam) primary antibody, diluted with BSA (diluted 1,000 times to 1 μM), was added to each slide drop and placed in a wet box, which was incubated at 4°C overnight. Next, the goat anti-rat IgG H&L Alexa Fluor 488-conjugated (ab150157, Abcam), donkey anti-rabbit IgG H&L-conjugated (Alexa Fluor® 488) (ab150073, Abcam), goat anti-mouse IgG H&L-conjugated (Alexa Fluor 594) (ab150116, Abcam), and donkey anti-rabbit IgG H&L Alexa Fluor 647-conjugated (ab150075, Abcam) secondary antibodies were used for labeling. The cells were imaged with a laser scanning confocal microscope. The average fluorescence intensity of 20 representative cells was obtained for quantitative analysis.

For cultured cells and culture medium, proteins were extracted using sodium dodecyl sulfate lysis buffer (2% sodium dodecyl sulfate, 10% glycerol, 0.1 mM dithiothreitol, and 0.2 M Tris–HCl, pH 6.8). For brain tissues, proteins were extracted using RIPA buffer. Protein samples (50ug) were resolved by SDS-PAGE and detected with following antibodies: STAT3 (9139, CST), Phospho-STAT3 (Tyr705) (9145, CST), IDE (ab32216, abcam), MMP2 (87809, CST), NEP (ab255609, abcam), PSD-95 (3450, CST), Synapsin-1 (5297, CST), GluR1 (AB1504, Milllipore), and Synaptophysin (36406, CST). The protein bands were quantified by densitometry analysis using Image J software and the intensity of each target protein was normalized by α–Tubulin intensity.