Bca method

Western blot analysis was performed routinely. Cells were seeded in culture plates under different conditions. All cells were washed with PBS buffer and lysed by lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were measured using the BCA method (Invitrogen Inc., United States). Equal quantities (30 μg) of proteins were loaded and separated on 10% SDS–polyacrylamide gels and then transferred onto PVDF membranes. The cells were blocked by TBST containing 5% skim milk at RT for 1 h and washed three times. The cells were incubated with primary antibodies for blots at 4°C overnight. All the primary antibodies used are listed as follows:

p-PI3K (Abcam, United States, Ab278545)

p-AKT (Abcam, United States, Ab38449)

p-mTOR (Abcam, United States, Ab109268)

GPX4 (EterLife, Birmingham, UK, EL604340)

xCT (EterLife, Birmingham, UK, EL611069)

4HNE (EterLife, Birmingham, UK, EL908789)

ATM (Abcam, United States, Ab201022)

Runx2 (Abcam, United States, Ab236639)

BMP2 (EterLife, Birmingham, UK, EL601094)

The blots were then washed in TBST and incubated with secondary antibodies at RT for 2 h. The blots were washed in TBST. The immunoreactive bands were visualized by chemiluminescence using enhanced chemiluminescence plus (GE Healthcare). GAPDH was used as an internal marker. The protein level was expressed as the ratio of the target band gray value to that of GADPH.

RIPA buffer added to HT22 cells (Invitrogen; USA) to collect the total protein; then the protein concentrations were determined assessed using the BCA method (Invitrogen, USA). The protein samples were mixed with a 5x loading buffer and denatured at the boil. The proteins were separated by 15% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Next, the membranes were incubated with primary antibodies (Bcl-2, Bax, caspase-3, BDNF, NGF, SIRT1, Wnt1, β-catenin, Shh, Patch, and GAPDH) at 4°C overnight. Then, the next day, the membranes were washed with PBS and incubated with secondary antibodies for 1 h. Finally, the protein bands were measured using the ImageJ software. The data were collected from at least three independent experiments.

After the transfected cells were washed twice in 6-well plates with PBS, 150 μL RIPA III lysis solution (Sangon Biotech, Shanghai, China) was added and lysed on ice for 15 min. The cells were centrifuged at 4 °C for 2 min at 12,000× g rpm, and the supernatant was transferred to a new centrifuge tube. The protein concentration was determined by BCA method (Invitrogen, Carlsbad, CA, USA), and then the protein samples were boiled in 5 × SDS protein loading buffer (Solarbio, Beijing, China) at 100 °C for 10 min. Two micrograms protein from each cell extract were resolved by 10% Acr-Bis SDS-PAGE and transferred to polyvinylidene difluoride membranes (PVDF, Millipore). Blots were detected by incubating with the antibodies BMF (Sangon Biotech, NO. D162765, 1:1000 dilution), P-AKT (Cst, #4060S, 1:1000 dilution), AKT (Cst, #4685S, 1:1000 dilution), BCL2 (Cst, #4223, 1:1000 dilution), P-AMPK (Cst, #4184, 1:1000 dilution), AMPK (Cst, #5831, 1:1000 dilution) or β-Actin at 4 °C overnight. Immunoreactive bands were then probed with appropriate horseradish peroxidase (HRP)-coupled secondary antibodies, such as goat anti-rabbit Ig G-HRP or goat anti-mouse Ig G (H + L)/HRP at room temperature for 2 h, and then protein bands were detected with chemiluminescent HRP substrate (Invitrogen, Carlsbad, CA, USA).