Quantity one 4 2 0

The cells were washed three times with PBS and suspended in ice-cold lysis buffer (Bio-Rad, Hercules, CA, USA). The lysates were separated by 12% SDS-PAGE, and the proteins of equal quantity were transferred to nitrocellulose membranes. Membranes were blocked with Tris-buffered saline with Tween-20 buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing 5% nonfat milk (Yili, China) and incubated overnight at 4°C with collagen II, collagen X (Abcam, Cambridge, MA, USA), aggrecan, Bcl-2, Bax, caspase-3 (Santa Cruz, Dallas, TX, USA), p21, p27, CDK2, CDK4 (Cell Signaling Technology, Danvers, MA, USA), p-ERK1/2, and ERK1/2 (Abcam, Cambridge, MA, USA) primary antibodies. The following day, the membranes were washed and incubated with the corresponding secondary antibodies at room temperature (22-28°C) for 1 h. An enhanced chemiluminescence detection system (Thermo Scientific, MA, USA) was finally used to determine the emission of the membrane. The experiments were performed in three independent replicates. The GAPDH expression level was used as an internal control. The Western blot results were quantified with an image analyzer (Quantity One-4,2,0, Bio-Rad, Hercules, CA) and were normalized to GAPDH immunostaining. The relative protein expression was compared with the control group.

After chemical treatment, cells were lysed with RIPA buffer supplemented with phosphatase inhibitor. Cell lysates were subjected to immunoblotting with antibodies; p-Akt (Ser473) (1:500, Cell Signaling Technology, Danvers, MA, USA), Akt (1:1000, Cell Signaling), p-GSK-3β (Ser9) and GSK-3β (1:1000, Santa Cruz Biotechnology, Dallas, Texas, USA), cytochrome c (1:500, Santa Cruz Biotech), PARP (1:1000, Santa Cruz Biotech), caspase-3 (1:1000, Cell Signaling), p-Tau (Ser396) and Tau (1:1000, Invitrogen), and GAPDH (1:1000, Santa Cruz Biotechnology). To evaluate cytosolic cytochrome c levels, cell pellets were fractionized using the Qproteome cell compartment kit (Qiagen, Germantown, MD, USA). The reactive bands were detected by ECL (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and quantified with an image analyzer (Bio-Rad, Quantity One-4, 2, 0). The same membranes were probed for GAPDH as an internal control [44 ].

Fresh spinal cord tissues were dissolved and homogenized in RIPA lysis buffer containing 1% protease inhibitor phenylmethanesulfonyl fluoride (PMSF) (Sangon Biotech; China). Proteins were loaded and separated in SDS-PAGE gel and then transferred to polyvinylidene fluoride membranes. After being blocked, membranes were incubated at 4°C overnight with the following antibodies: anti-Sirt1 (1 : 1000; Santa Cruz; USA), anti-PGC1-α (1 : 200; Santa Cruz; USA), anti-p53 (1 : 1000; Cell Signaling; USA), anti-acetyl-p53 (1 : 1000; Millipore; USA), anti-Bax (1 : 1000; Cell Signaling; USA), anti-Bcl-2 (1 : 1000; Cell Signaling; USA), anti-caspase-3 (1 : 1000; Cell Signaling; USA), anti-Cytochrome C (Cyt C) (1 : 4000; Abcam; USA), and anti-PARP (1 : 1000; Santa Cruz; USA). After being washed thoroughly, the membranes were incubated with the appropriate secondary antibody for 2 hours. Protein bands were visualized by ECL (Pierce; USA) and an image analyzer (Quantity One-4.2.0; Bio-Rad; USA) was used to quantify the density of interested bands.