Criterion cell system

Using published protocols [107 (link),110 (link),111 (link)], MCF7 and MCF10 cells were homogenised in RIPA lysis buffer, supplemented with a cocktail of protease inhibitors (cOmplete; Roche Diagnostics, Milano, Italy). Equal amounts of proteins were separated by 4–20% SDS-polyacrylamide gel electrophoresis (Criterion TGX Stain-free precast gels and Criterion Cell system; Bio-Rad, Hercules, CA, USA) and transferred onto nitrocellulose membrane using a Bio-Rad Trans-Blot Turbo System. When indicated, the membranes were probed using the rabbit monoclonal anti-cleaved caspase 7 and anti-XIAP (Cell Signaling Technology) primary antibodies. After the incubation with the appropriate horseradish-peroxidase-conjugated secondary antibody (Cell Signaling Technology), bands were visualised using the Clarity Western ECL substrate with a ChemiDoc MP imaging system (Bio-Rad). To monitor for potential artefacts in loading and transfer among samples in different lanes, the blots were routinely treated with the Restore Western Blot Stripping Buffer (ThermoFisher Scientific) and re-probed with the goat anti-Lactate dehydrogenase (LDH)-A (Santa Cruz Biotechnology) and the mouse anti-vinculin primary antibodies. The stain-free gel was used as loading control as well. When appropriated, bands were quantified for densitometry using the Bio-Rad Image Lab software.

Hydrolysates collected from the digestion simulation were loaded onto Novex™ NuPAGE™ 10% Bis-Tris Midi Protein Gels (Invitrogen^TM, Thermo Fisher Scientific, Auckland, New Zealand) by 40 µg proteins per well and separated at ambient temperature in a Bio-Rad Criterion cell system at 150 V using PowerPac™ HC High-Current Power Supply (Bio-Rad^® Laboratories, Hercules, CA, USA). An 8-µl aliquot of Novex™ Sharp Pre-stained protein standard (Invitrogen, UK) was used to determine molecular weight (MW) of different protein sizes from 3.5 to 260 kDa. Following electrophoresis, gels were stained in a SimplyBlue SafeStain (Invitrogen^TM) for 4 h. Stained gels were then washed with distilled water and images were captured with a GS900 calibrated densitometer scanner (Bio-Rad^® Laboratories). The gels were loaded following the order which enabled the time-course of digestion (0, 2, 10, 60, 120 and 240 min) to be visualised and for dry- and wet-ageing treatments to be compared.

Protein lysates from cortical neurons, 3xTg-AD mice and human brains were separated by SDS-PAGE using 7.5% Tris-Glycine polyacrylamide gels (Bio-Rad). Electrophoresis was conducted in a Tris-Glycine buffer (25 mM Tris, pH 8.3, 192 mM glycine, 0.1% SDS in dH₂O) by using the Criterion cell system (Bio-Rad). Blots were developed with rabbit anti-NR2B (1:1000, Millipore, #AB1557P), rabbit anti-pSer¹³⁰³NR2B (1:1000, Millipore, #07-398), rabbit anti-NR2A (1:1000, Millipore, #AB1555P), rabbit anti-PSD95 (1:500, abcam, #ab18258), mouse anti-synaptophysin (1:500, Millipore, #MAB329), rabbit antiphosho PKC (1:1000, Cell Signaling, #9371), rabbit anti-PKC (1:1000, abcam, #ab179521) and rabbit anti-β-actin (1:5000; Sigma-Aldrich, #A2066). Secondary antibodies conjugated with horseradish peroxidase (HRP) were purchased from Sigma (1:5000, sheep antimouse-HRP #A6782, goat antirabbit-#HRP A6154).

Cultured MDMs were removed by non-enzymatic cell dissociation solution (Sigma, Saint Louis, Missouri, USA), counted by the automated cell counter ADAM-MC (Digital Bio) and used for protein extraction by RIPA buffer (Sigma, Saint Louis, MO, USA). Extracted proteins were stored at −80 °C for further analyses. For WB analyses, samples from 3 HeteroAB subjects were sub-pooled (50 μg per pool). Equal amounts of proteins were separated by 4–20% SDS-polyacrylamide gel electrophoresis (Criterion TGX Stain-free precast gels and Criterion Cell system; Bio-Rad) and transferred onto nitrocellulose membrane using a Bio-Rad Trans-Blot Turbo System. Membranes were probed using a 1:1000 dilution of primary antibody goat anti-ERAP1 (AF2334; R&D Systems, Minneapolis, MN, USA) goat anti-ERAP2 polyclonal antibody (AF3830; R&D Systems, Minneapolis, MN, USA), rabbit anti-GAPDH polyclonal (VPA00187); Bio-Rad, Hercules, CA, USA] and a 1:10,000 dilution of secondary antibody conjugated with alkaline phosphatase anti-goat IgG (A4187; Sigma; goat anti-rabbit (STAR208P); Bio-Rad]. Membranes were incubated with the appropriate antibody and, after being excited using the Clarity Western ECL substrate, bands were visualized with a ChemiDoc MP imaging system (Bio-Rad) and quantified for densitometry with the Bio-Rad Image Lab software.