Ripa buffer

Frozen mouse dorsal skin samples were homogenized (at 10 mL/g) in RIPA buffer (Fujifilm, Tokyo, Japan) supplemented with a protease-inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and phosphatase-inhibitor cocktail (Nacalai Tesque). Lysates were centrifuged at 15,000 × g for 20 min at 4 °C, and then 10-μg aliquots of protein were separated on 10–20% SDS–polyacrylamide gels, transferred onto polyvinylidene difluoride membranes (Immobilon-P; Merck Millipore, Burlington, MA, USA), and immunoblotted with the following primary antibodies: rabbit anti-MMP-9 polyclonal antibody (1:1000; Gene Tex, Hsinchu, Taiwan), rabbit anti-COX-2 polyclonal antibody (1:1000; Cell Signaling Technology (CST), Danvers, MA, USA), rabbit anti-catalase polyclonal antibody (1:1000; Gene Tex), rabbit anti-SOD1 polyclonal antibody (1:1000; Gene Tex), rabbit anti-SOD2 polyclonal antibody (1:1000; Gene Tex), and mouse anti-β-actin monoclonal antibody (1:1000; CST). The secondary antibodies used were HRP-conjugated goat anti-rabbit IgG (1:5000; CST) or goat anti-mouse IgG (1:5000; CST). Immunoreactive bands were visualized using an Amersham Imager 680 (GE Healthcare, Chicago, IL, USA). Band intensity was measured using Fiji.

The level of Aβ_1–42 in cerebral cortex was measured by enzyme-linked immunosorbent assay (ELISA) using a human/rat β amyloid (42) ELISA kit Wako, High-Sensitive (Wako Pure Chemical Industries, Ltd.). Samples were prepared as described by Hosoda et al. [32 (link)] and Borchelt et al. [33 (link)] with minor modifications. Approximately 100 mg of cerebral cortex was homogenized with a Physcotron homogenizer (Microtec Co., Ltd., Tokyo, Japan) in 0.7 mL of 70% formic acid and centrifuged at 100,000× g for 1 h to obtain soluble and insoluble fractions. The supernatant was neutralized by diluting it 20-fold in 1 M Tris base. The levels of soluble and insoluble Aβ_1–42 were measured according to the manufacturer’s instructions. To assess oxidative stress, the level of malondialdehyde (MDA) was measured by thiobarbituric acid reactive species (TBARS) assay using an MDA ELISA kit (Japan Institute for the Control of Aging, NIKKEN SEIL Co., Ltd., Shizuoka, Japan). Approximately 100 mg of cerebral cortex was homogenized with a Physcotron homogenizer (Microtec Co., Ltd.) in 250 µL of RIPA buffer (Wako Pure Chemical Industries, Ltd.). Homogenates were centrifuged at 1600× g for 10 min, and the supernatants were used for the TBARS assay according to the manufacturer’s procedure.

Western blotting was performed as described previously with some modifications^{18 (link)}. Briefly, cells were lysed with RIPA buffer (Wako) containing a protease inhibitor mixture (Roche Diagnostics, Basel, Switzerland). Equal amounts of protein (10 μg) were electrophoresed using 10% sodium dodecyl sulphate–polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes, washed with Tris-buffered saline containing 0.05% Triton X-100, and incubated with BlockingOne solution (Nacalai Tesque, Kyoto, Japan) for 60 min. Anti-p62 (1:1,000; MBL) and anti-ubiquitin (1:1,000; Santa Cruz Biotechnology) antibodies were used as the primary antibodies. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies (Promega, Madison, WI, USA) were used as the secondary antibodies. As a control, β-actin was detected with anti-β-actin pAb-HRP-DirecT (1:2000; MBL). Blots were visualized using Chemi-Lumi One L (Nacalai Tesque). Stained membranes were scanned with ImageQuant LAS 4000 (GE Healthcare, Menlo Park, California, USA). Quantification was performed using ImageJ software (https://imagej.nih.gov/ij/).