Gapdh g9545

Western blotting was carried out as described previously [5 (link)].
Antibody dilutions: Carbonic anhydrase II ab6621 (Abcam) 1:7000 dilution in 3% (w/v) BSA in TBS-T; NADH dehydrogenase flavoprotein 2 ARP57510-PO50 (Cambridge Bioscience) 1:5000 dilution in 3% (w/v) BSA in TBS-T; Beta-actin ab8227 (Abcam) 1:5000 dilution in 3% (w/v) BSA in TBS-T; Carbonic anhydrase III AP7633a (ABGENT) 1:2500 dilution in 3% (w/v) BSA in TBS-T, GAPDH G9545 (SIGMA) 1:5000 dilution in 3% (w/v) BSA in TBS-T and COXIV (ab16056) 1:5000 dilution in 3% (w/v) BSA in TBS-T. Brain mitochondrial samples were normalised to beta-actin level. The average of four samples for each condition (old and young) were plotted showing the mean +/− SEM. The muscle mitochondrial samples were normalised to GAPDH level. The average of the four samples for each condition (old and young) were plotted showing the mean +/− SEM. The retina mitochondrial samples were normalised to COXIV level. The average of the six samples for each condition (young and old) were plotted showing the mean +/− SEM. Statistical analyses (unpaired t-tests with Welch's correction) were carried out in GraphPad Prism.

The following antibodies were used: CD4–14–0041, CD8–14–0081, and Ly6G—14–5931, all rat monoclonal (eBioscience); IL1B—8689, rabbit polyclonal (Cell Signaling Technology); F4/80—ab74383 and collagen type-Ia—ab34710, both rabbit polyclonal (Abcam); CD68—MCA1957GA and CD206—MCA2235GA, both rat monoclonal (AbD Serotec); CD163—sc-33560, rabbit polyclonal (Santa Cruz Biotechnology); dystrophin—MANDRA1, mouse monoclonal, and myogenin—FD5, rat monoclonal (Developmental Studies Hybridoma Bank); LC3II—L7543 and actin—A2066, both rabbit polyclonal, and GAPDH—G9545 (Sigma-Aldrich); collagen type-IV—AB769, goat polyclonal (Chemicon); P2X7–177 003, rabbit polyclonal (Synaptic Systems); β-tubulin—IMG-5810A, rabbit polyclonal (Imgenex); dystrophin—2166, rabbit polyclonal, was a kind gift from D. J. Blake, Cardiff University.

Histone Acetyltransferase p300 inhibitor, C646 (382113), protease inhibitor cocktail (P2714), dimethyl sulfoxide (DMSO, D8418) and propidium iodide (PI, P4170) were purchased from Sigma-Aldrich, USA and romidepsin (FK228, Depsipeptide) from Seleck Biochem, USA. Lipofectamine 2000 (11668), Alexa fluor 488 goat anti-rabbit (A11034), Phalloidin 568 (A12380), Phalloidin 633 (A22284) and Hoechst 33258 (H3569) were purchased from Thermo Fisher Scientific, USA. GFP (ab290), RFP (ab62341) Cyclin B1 (ab32053), p21 (ab109520) and p300 (ab10485) antibodies were obtained from Abcam, USA, HDAC1 (sc-8410), E2F1 (C-20- sc-193) and Cyclin A (sc-271682) from Santacrutz Biotechnology Inc, USA. PCNA (2586), pAkt Ser437 (9271), Akt (9272) and acetyl p53 Lys382 (2828) from Cell Signaling Technology, USA. Api5 (PAB7951) from Abnova, Taiwan, acetyl lysine (05-515) from Merck Millipore, USA, Hoechest 3,258 (H3569) was purchased from Molecular Probes, Thermo Fisher Scientific, USA and GAPDH (G9545) from Sigma-Aldrich, USA.

Cellular pellets were lysed in RIPA buffer: 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA and 1% Triton-X100 and protease inhibitors (Sigma-Aldrich Inc., Saint Louis, MO). Samples were resolved on 4–12% SDS-PAGE gels using a mini-gel apparatus (Bio-Rad Laboratories, Richmond, CA) and transferred to Hybond-C extra nitrocellulose (Amersham Pharmacia Biotech, Piscataway, NJ). Membrane was blocked for 1 h with 5% non-fat dry milk in TBS containing 0.05% Tween-20 and incubated over night with primary antibody. The primary antibodies used are: pAKT S473 (4058) and pERK T202/Y204 (9101) (all from Cell Signaling Technology, Danvers, MA) and GAPDH (G9545) (Sigma-Aldrich Inc., Saint Louis, MO). Washed filters were then incubated for 45 min with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ) and visualized by using an enhanced chemioluminescence detection system (ChemiDoc XRS+ (Bio-rad) with Image Lab software).