Anti nqo1

After the different treatments, mixed glial cultures and mouse hippocampi were lysed in ice-cold lysis buffer (1% Nonidet P-40, 10% glycerol, 137 mM NaCl, 20 mM Tris–HCl, pH 7.5, 1 g/mL leupeptin, 1 mM PMSF, 20 mM NaF, 1 mM sodium pyrophosphate, and 1 mM Na₃VO₄). Proteins (30 µg) from cell lysates were separated via SDS-PAGE and transferred to Immobilon-P membranes (Millipore Corp., Billerica, MA, USA). Membranes were incubated with anti-NLRP3 (1:1000; AdipoGen, San Diego, CA, USA), anti-NOX4 (1:1000; R&D Systems, Minneapolis, MN, USA), anti-NQO1 (1:1000; Cell Signaling Technology, Danvers, MA, USA) or anti-βactin (1:50,000; Sigma-Aldrich, Madrid, Spain). Appropriate peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology, Dallas, TX, USA) were used for protein detection via enhanced chemiluminescence on the ImageQuant LAS 4000 min. Different band intensities corresponding to immunoblot detection of protein samples were quantified using the Scion Image program. Immunoblots corresponded to a representative experiment that was repeated three or four times with similar results.

Mice were perfused with 4% paraformaldehyde in 0.1% phosphate buffer and post-fixed for 24 h in the same fixative. The head tissue, including the cochlea, OE, and olfactory bulb (OB), was decalcified with 10% EDTA solution, pH 7.0, and embedded in paraffin. Coronal sections were cut at 4 μm thickness and mounted on silane-coated slides. Deparaffinized sections were autoclaved at 121 °C for 20 min in Target Retrieval Solution (S1700; Dako) for antigen retrieval. Immunohistochemistry was performed with the following antibodies: anti-olfactory marker protein (OMP, goat polyclonal, 1:2000 dilution; Wako Chemicals), anti-NQO1 (rabbit polyclonal, 1:300; Cell Signaling Technology), anti-activated caspase-3 (rabbit polyclonal, 1:300; Cell Signaling Technology), anti-Ki67 (mouse monoclonal, 1:300; BD Biosciences), anti-c-fos (rabbit polyclonal, 1:50; Santa Cruz Biotechnology), anti-MnSOD (rabbit monoclonal, 1:100; Epitomics Inc.), and anti-8-hydroxy-2′-deoxyguanosine (8-OHdG, goat polyclonal antibody, 1:100; Alpha Diagnostic International Inc.). Immunoreactivity was detected using the following antibodies in the Histofine Simple StainMAX-PO secondary antibody system (Nichirei) according to the manufacturers’ instructions, donkey anti-goat Alexa Fluor 488 (1:100; Invitrogen), and donkey anti-rabbit Alexa Fluor 594 (1:100; Invitrogen), incubated for 1 h at room temperature.

Cells were harvested and lysed and protein content of cell lysates was measured as described previously [26 (link)] Whole cell lysates were separated by SDS-PAGE (in 10% or 15% SDS-polyacrilamide gel). SDS-PAGE and Western Blot analysis was done according to our previous study [26 (link)]The following antibodies were applied: anti-NQO1 (Cell Signaling, A180), anti-CyclinD1 (Santa Cruz, A-12), anti-p21 (Santa Cruz, C-19), anti-p27 (Santa Cruz, C-19), anti-p15-INK4b (proteintech, 12877-1-AP), anti-p16-INK4A (proteintech, 10883-1-AP), anti-PERK (Cell Signaling, 3192S), anti-P-eIF2α (Cell Signaling, 9722S9), anti-P-eIF2α (Ser51) (Cell Signaling, 9721L), and anti-GAPDH (Santa Cruz, 6C5) and HRP conjugated secondary antibodies (SantaCruz, sc-2020 and Cell Signaling, 7074S, 7076S).