Geldox xr

The protein expression of Ftmt was analyzed by Western blotting as previously described [17 (link),18 ]. Briefly, the brain cortex protein from injured area was extracted using RIPA buffer (Beyotime, Jiangsu, China) containing 1% protease inhibitor cocktail (Roche, Mannheim, Germany). The protein concentration was determined by Protein Assay Kit (Thermo, MA, U.S.A.). Equal protein of each sample was separated on 8% Tris/HCl polyacrylamide gel, and then transferred to a polyvinylidene difluoride membrane (Millipore, MA, U.S.A.). After blocking in 5% nonfat milk for 1 h, the membranes were incubated with primary antibodies against Ftmt (1:500; Sigma, SAB2700108, MO, U.S.A.) and GAPDH (1:1000; Santa Cruz, sc-25788, CA, U.S.A.) at 4°C overnight. At the end of incubation period, membranes were then incubated with secondary antibody (1:1000; goat anti-rabbit, Millipore, AP307P) conjugated to horseradish peroxidase (HRP) for 1 h at room temperature. Bands were visualized using an enhanced chemiluminescence system (Pierce Biotech, IL, U.S.A.). The images were captured by the gel imager (GelDox XR+, Bio-Rad, CA, U.S.A.) and the densitometry of each band was quantified by Image-pro software (Media Cybernetics, MD, U.S.A.).

Tumors were homogenized using the FastPrep-24 homogeniser (MP Biomedicals, CA, USA) and lysed in 1% (v/v) Triton X-100, 50 mM HEPES (pH 7.4) 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 100 mM NaF, 10 mM Na₄P₂O₇, 1 mM Na₃VO₄, 10% (v/v) glycerol, cOmplete^TM, EDTA-free Protease Inhibitor Cocktail and PhosSTOP^TM (Roche) at 4 °C. Cells were washed twice in ice cold PBS and lysed in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% TritonX-100, 0.5% sodium deoxycholate, 0.1% SDS) containing cOmplete^TM, EDTA-free Protease Inhibitor Cocktail and PhosSTOP^TM (Roche) at 4 °C prior to SDS-PAGE. Proteins were transferred to nitrocellulose and membranes incubated with antibodies to HO-1 (catalogue number 5853; 1:1000), p62 (catalogue number 5114; 1:1000), pErk1/2 (Thr202/Tyr204) (catalogue number 4370; 1:1000), Erk1/2 (catalogue number 4696; 1:1000), pJNK (Thr183/Tyr185) (catalogue number 9251; 1:1000), JNK (catalogue number 9252; 1:1000) (all Cell Signaling, MA, USA) or LC3B (catalogue number L7543; 1:3000; Sigma Aldrich, MO, USA) overnight at 4 °C. Membranes were washed thrice with TBST before incubation with HRP-conjugated secondary antibodies (Cell Signaling, MA, USA1:1000). Washes were repeated in TBST and bound antibodies detected by chemiluminescence using Clarity ECL Substrate (BioRad, Germany) on the BioRad Gel Dox XR+.

PCR amplification was conducted in 10 µl reaction mixtures, which contained 20 ng template DNA, 10 pmol of each forward and reverse primers, and the 2× Taq PCR Master Mix (Tiangen Biotech, Beijing, China). The reaction was carried out with a thermocycler (Biometra, Göttingen, Germany) using the following conditions: an initial denaturation step of 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, an appropriate annealing temperature of different primers (Table 1 and Supplemental Tables S2 and S3) for 30 s, 72°C for 2 min, and a final elongation step of 72°C for 10 min.
Amplified products were separated on a 1.5% agarose gel in 1×TAE buffer along with the DNA ladder DL2000 (Sangon, Shanghai, China) as a size marker. Next, the PCR products were visualized and photographed under a Gel Dox XR+ (Bio-Rad, Madison, CA, USA) photography system using ultraviolet light. Data were scored manually for band presence or absence and entered into a matrix for further analysis.