Hdac6

Sodiumdodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were performed as described previously 5 (link). The following antibodies were used: MAD2 (BD BioScience, San Jose, CA, USA), HDAC6 (Millipore, Billerica, MA, USA), Caspase 3, p21, poly (ADP-ribose) polymerase (PARP), CDK6 and p-Rb (Ser 807/811) (Cell Signalling, Danvers, MA, USA), β-actin and p16 (Santa Cruz Biotechnology, Dallas, Texas, USA), beta (β)-galactosidase (Abcam, Cambridge, UK).

Whole retinas were extracted from mice after removal of the lens and cornea and then lysed using a tissue lyser (JXFSTPRP‐24L; Jingxin, Shanghai, China), in cold lysis buffer containing Tris (50 × 10⁻³ m, PH 7.5), NaCl (150 × 10⁻³ m), EDTA (1 × 10⁻³ m), glycerin (3%), NP40 (1%), and complete protease inhibitor tablets (Roche, Basel, Switzerland). Lysates were cleared by centrifugation at 15 000 rpm for 20 min at 4 °C. Supernatants were added with SDS sample buffer (pH 6.8) containing urea (8 m) and subjected to 8% SDS‐PAGE. Proteins were transferred to polyvinylidene difluoride membranes (Millipore) and subjected to immunoblot analysis as described previously.^[7] Primary antibodies for immunoblotting were as follows: HDAC6 (07‐732; Millipore), pT845‐ASK1 (3765; Cell Signaling Technology), α‐tubulin (ab18251; Abcam), acetylated‐α‐tubulin (T6793; Sigma‐Aldrich), and β‐actin (A5316, Sigma‐Aldrich).

To measure the protein concentrations in the hippocampus, we homogenized the tissue in RIPA lysis buffer supplemented with a cocktail of protease and phosphatase inhibitors (Applygen, China) on ice. After centrifugation, the supernatant protein concentration was determined with a bicinchoninic acid (BCA) protein assay reagent kit (Thermo Pierce, USA). After determination of the protein concentration, protein samples were separated by SDS-PAGE and subsequently transferred to PVDF membranes (Bio-Rad, USA). Membranes were blocked with 5% nonfat milk in TBST (0.1% Tween-20 in TBS) and then incubated at 4 °C overnight with primary antibodies: HDAC2 (1:2000, Cell Signaling Technologies, USA), HDAC3 (1:2000, Santa Cruz, USA), HDAC4 (1:2000, Cell Signaling Technologies, USA), HDAC6 (1:4000, Merck Millipore, USA), Acetyl-H3 (1:2000, Merck Millipore, USA), Acetyl-H3K9 (1:2000, Merck Millipore, USA), Acetyl-H3K14 (1:2000, Merck Millipore, USA), BDNF (1:1000, Abcam, UK), c-Fos (1:2000, Merck Millipore, USA), and β-actin (1:2000, Santa Cruz, USA). After being rinsed with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:1000, Beyotime Institute of Biotechnology, China). Protein bands were visualized with an ECL detection system (Beyotime Institute of Biotechnology, China) and quantified with ImageJ software (NIH).

SDS-PAGE and western blotting were performed as described previously [58 (link)] with the following antibodies; acetylated α-tubulin, acetylated H3, (Cell Signaling, London, UK), WT-1, CK8, p53 (Abcam, Cambridge UK), HDAC6 (Merck, Hertfordshire, UK). β-actin and GAPDH (Sigma, Hertfordshire, UK) were used as loading controls. The levels of protein expression were assessed using a SuperSignal™ West Pico PLUS Chemiluminescent detection kit (ThermoFisher, Paisley, UK), and G:BOX imaging system (Syngene, Cambridge, UK).