Ipvh0010

The brain tissues and collected cells were stored at − 80 °C before use and homogenized to determine protein expression using the western blotting protocol described in our previous study [19 ]. The protein concentration was measured using the BCA Protein Assay Kit (P0010; Beyotime, China). Electrophoresis was performed using 10% or 12.5% SDS-polyacrylamide gels, and then proteins were electrotransferred to polyvinylidene fluoride membranes (IPVH0010; Millipore, Germany). The membranes were blocked with BSA or 5% non-fat milk and incubated with primary antibodies β-tubulin III (1:1000, T2200; Sigma-Aldrich), GFAP (1:10,000, ab53554; Abcam, UK), nestin (1:1000, ab6142; Abcam), ATN1 (1:500, orb213859; Biorbyt, UK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, 60004-1-Ig; Proteintech, USA) overnight at 4 °C. The membranes were incubated with second antibodies the next day for 2 h at room temperature, and then photographed using a GE Amersham Imager 600. Number of tissues was 5 per group and number of cells was 3 per group in western blot experiments. Images were analyzed using Image-Pro Plus 6.0 software.

The experiment was performed as previously described.¹⁴ First, RIPA buffer was prepared, and cells were lysed for 30 min on ice. Then, the cell lysates were collected and centrifuged at 14,000 rpm for 30 min at 4°C. After that, the protein concentrations were measured, and equal amounts of proteins were mixed with loading buffer. The proteins were separated via electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, IPVH0010). Then the membranes were blocked with 5% non‐fat milk solution for 1 h. Thereafter, the membranes were incubated with primary antibodies followed by secondary antibodies. Finally, protein levels were detected using Chemiluminescent HRP Substrate (Millipore, WBKLS0500) and analyzed with ImageJ.
The following antibodies were used for western blotting: anti‐Osterix (Abcam, ab209484, 1:800); anti‐OCN (Abcam, ab93876, 1:800); anti‐AGO2 (Abcam, ab186733, 1:1500); anti‐RUNX2 (Cell Signaling Technology, 12556S, 1:1000); anti‐Collagen I (ColI; Abcam, ab260043, 1:1500); anti‐IRF2 (Abcam, ab124744, 1:2000); anti‐YY1 (Abcam, ab109228, 1:2000); anti‐GAPDH (CWBIO, CW0100M, 1:3000); and HRP‐conjugated secondary antibodies (Boster, 1:5000).

The cell protein was extracted by using RIPA buffer followed with centrifuge at 14,000 rpm for 30 min at 4 °C. Then, the protein concentrations were detected using a BCA Protein Assay Kit (CWBIO, CW0014S) and equal amount of protein were mixed with sodium dodecyl sulfate (SDS) loading buffer. The mixtures were boiled for 10 min and stored at −80°Cor used directly. For western blot assay, the SDS-page gels (Beyotime, P0012A) were prepared according to the instruction and the proteins were separated via electrophoresis. Then the proteins were transferred to polyvinylidene fluoride membranes (Millipore, IPVH0010) and incubated with primary antibodies at 4 °C overnight after being blocked by 5% non-fat milk. On the second day, the membranes were washed and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The protein signals were detected by Immobilon Western HRP Substrate (Millipore, WBKLS0500) and the gray values were analyzed by image J.

Total protein content was extracted from cells or tissues using RIPA lysis buffer (Beyotime, P0013B), supplemented with a mixture of protease inhibitors (Roche, 11697498001). The protein concentration was determined using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, 23225), following the manufacturer’s protocols. The protein concentration was adjusted to 1 mg/ml using 5 × SDS-PAGE sample loading buffer (Beyotime, p0015), based on the BCA standard curve. Proteins were then separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in 1 × running buffer (14.4 g glycine, 2.9 g Tris base, and 1 g SDS per 1 L dd water). The proteins were transferred to a 0.45 μm polyvinylidene fluoride (PVDF) membrane (Millipore, IPVH0010) in 1 × transfer buffer (7.2 g glycine, 1.45 g Tris base, and 20% methanol per 1 L dd water) and then blocked using 5% skimmed milk. The membranes were then incubated with the corresponding primary and secondary antibodies, and the target protein bands were visualized using an Enhanced Chemiluminescence kit (Thermo Fisher Scientific, 32132).

The tissue was homogenized in a radioimmunoprecipitation assay lysis buffer (P0013B, Beyotime Biotechnology) with a 1.0% protease inhibitor cocktail (P8349, Sigma‐Aldrich) or by the Nuclear and Cytoplasmic and Protein Extraction Kit (P0028, Beyotime) to extract cytoplasmic‐nucleoprotein, and then incubated on ice for 30 min. Protein concentrations were determined using a BCA Kit (P0010, Beyotime). We used 6% or 10% SDS‐polyacrylamide gels for electrophoresis and then electrotransferred the proteins to polyvinylidene difluoride membranes (IPVH0010, Millipore). The PVDF membranes were blocked with 5% skimmed milk for 2 h, followed by incubation with primary antibodies: ACSL4 (1:2000, ab155282, Abcam), GPX4 (1:4000, ab125066, Abcam), PTGS2 (1:600, 66351‐1‐Ig, Proteintech Biotechnology), SLC7A11 (1:1000, ab175186, Abcam), FTH1 (1:1000, #4393, Cell Signaling Technology), 15LO2 (1:250, sc‐376795, Santa Cruz), PEBP1 (1:1000, ab76582, Abcam), p‐PEBP1 (1:1000, ab75971, Abcam), p‐ATM (1:750, sc‐47739, Santa Cruz), ATM (1:3000, 67586‐1‐Ig, Proteintech), GAPDH (1:8000, 60014‐1‐Ig, Proteintech) and Histone‐H3 (1:1000, 17168‐1‐AP, Proteintech), respectively. Antibodies against NeuN (1:1000, #24307, CST) and GAPDH were also used in P0 hippocampal samples. Any protein band was measured by enhanced chemiluminescence and quantified using ImageJ software (NIH Image, Bethesda, USA).