Anti rabbit chip

Hippocampal slices were rinsed with PBS and then homogenized on ice in RIPA lysis buffer containing 1% TritonX-100, 1% DOC, 1% NP-40, NaCl 150 mM, TrisHCl 10 mM, EDTA 5 mM, SDS 0.1%, PMSF (10 mg/mL), pepstatin (1 mg/mL), aprotinin (2 mg/mL), leupeptin (2 mg/mL), NaF (22 mg/mL) and sodium ortovanadate (92 mg/mL). Lysates were centrifuged at 12,000 g for 15 min at 4°C. Protein concentration in the supernatant was determined with by Lowry protein assay. In a 10% SDS–polyacrylamide gel, 30 μg of protein was applied per lane for electrophoresis. After that, gel was transferred to nitrocellulose membranes (Bio-Rad) and processed for western blotting. First, membrane was blocked with 5% milk in T-TBS buffer (0.1% Tween in 20 mM Tris-HCl/137 mM NaCl; pH 7.3), then overnight with primary antibodies: anti-goat BIP/GRP78 (1:500, Santa Cruz); anti-rabbit phosphorylated eIF2-α (1:1000, Bioscience anti-rabbit eIF2-α (1:1000, Santa Cruz); anti-rabbit CHIP (1:1000, Santa Cruz) or anti-rabbit ERK-2 (1:2000, Santa Cruz). Washed membranes were incubated with an HRP-conjugated secondary anti-antibody for 1 h and revealed with the ECL Western Blotting Analysis reagent (Amersham Biosciences). Optical density on the blots was measured with ImageJ Software.

Hippocampal slices not treated with PI were fixed with PF 4% for 2 h and washed in PBS. After that, they were removed from the membrane inserts and placed in 24 well plates where they were permeabilized with 1% Triton X-100 in PBS for 2 h. Free floating slices were then incubated with 1% BSA in PBS for 2 h and incubated with primary antibodies in 1% BSA overnight at 37°C. Primary antibodies used include anti-rabbit CHOP/GADD153 (1:100; Santa Cruz) anti-mouse TUJ-1 (1:100; Sigma), anti-rabbit CHIP (1:100; Santa Cruz), anti-rabbit cleaved caspase-3 (1:100; Cell Signaling), anti-rabbit p53 (1:100, Santa Cruz). Following washes with PBS, tissues were incubated for 1 h at room temperature with Alexa Fluor 488-conjugated goat anti-rabbit, Alexa Fluor 555-conjugated goat anti-mouse antibodies (Invitrogen) diluted in PBS (1:200) plus TO-PRO3 (1:1000, Sigma) for nuclear staining. Tissues were then washed in PBS and mounted with N-propylgallate.
Slices were examined in a confocal microscope (Zeiss, LSM 510). Cells with condensed chromatin were counted in CA1 at 40x of magnification, in three distinct fields for each slice. Values represent mean percentages for slices under various treatments.

Dulbecco’s modified Eagle’s medium (DMEM)/Nutrient Mixture F-12 Ham, APAP, l-glutamine, l-ascorbic acid, anti-GAPDH antibody and Triton^TM X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Collagenase, insulin human recombinant and cycloheximide were purchased from Wako Pure Chemical Industries (Osaka, Japan). Soybean trypsin inhibitor powder, HEPES buffer (1 M) and Dynabeads^® Protein G were purchased from Thermo Fisher Scientific (Gibco^®; Waltham, MA, USA). 2-Mercaptoethanol, dexamethasone, Nonidet^® P-40 and polyoxyethylene sorbitan monolaurate were purchased from Nacalai tesque (Kyoto, Japan). MG132 (Z-Leu-Leu-Leu-H aldehyde) was purchased from Peptide Institute (Osaka, Japan). Proteasome substrates II, III and VI, and Fluorogenic and anti-rabbit CYP3A1 antibodies were purchased from Merck Millipore (Calbiochem^®; Darmstadt, Germany). Penicillin G potassium and streptomycin were purchased from Meiji Seika Pharma (Tokyo, Japan). Anti-mouse CYP3A1, anti-rabbit CHIP, anti-rabbit gp78-2, anti-rabbit Nrf2 and anti-mouse Ub antibodies were purchased from Santacruz Biotechnology (Dallas, Texas, USA). P450-Glo^TM CYP3A assay with luciferin-IPA was purchased from Promega (Fitchburg, WI, USA).