A19056

The protein was lysed using RIPA lysis buffer, and BCA method was adopted to determine the protein concentration. The precast gel was removed from the refrigerator at 4°C and put into the electrophoresis tank. Subsequently, 500 μg of total protein was added to each sample and mixed by the additional of 5× SDS loading buffer at 4:1 ratio. After mixing, the protein concentration was set at 3.3 μg/μL. The metal bath was heated at 100°C for 6 min to denature the protein. The denatured total protein was taken 60 μg for loading. After running at 80 V through the concentrated glue, the voltage was adjusted to 120 V, and waited until the bromophenol blue reached the bottom of the rubber sheet without spilt out. After transferring the membrane, 5% skimmed milk blocking solution was supplied for blocking at room temperature for 1 h. Primary antibody was added for incubation overnight at 4°C, washed 3 times with TBST, 10 min each time, added secondary antibody and incubated at room temperature for 1 h, washed with TBST 3 times, 10 min each time, and exposed using ECL exposure liquid. The antibodies used were: FEZ1 (A15362, abclonal, China), 1:500; SCOC (PH309356, ORIGEN, United States), 1:500; ULK1 (A8529, abclonal, China), 1:500; NBR1 (A3949, abclonal, China), 1:500; GAPDH (A19056, abclonal, China), 1:2000; and secondary antibody (AS014, abclonal, China), 1:1000.

BCA kit (P0012, Beyotime, China) was used to quantify the extracted protein. After quantification, it was then electrophoresed in 10% SDS-PAGE gel and transferred to a PVDF membrane (Millipore, USA). After blocking with 5% skim milk, the primary antibody was incubated at 4 °C overnight, and the secondary antibody was incubated at room temperature for 1 h. ECL kit (Tanon, China, 185001) and chemiluminescence instrument (Tanon, China) were used for imaging. The primary antibodies against were listed as follows: MMP13 (18165-1-AP; 1:2000; Proteintech), MMP3 (A11418; 1:1000; Abclonal), Col2a1 (ab307674; 1:1000; Abcam), ADAMTS5 (bs-3573R; 1:1000; Bioss), GPX4 (67763-1-Ig; 1:1000; Proteintech), Nrf2 (ab307026; 1:5000; Abcam), P53 (ab26; 1:500; Abcam), SLC7A11 (ab175186; 1:5000; Abcam), NF-κB p65 (8242T; 1:1000; CST), p-NF-κB p65 (3035; 1:1000; CST), and GAPDH (A19056; 1:10000; Abclonal).

Total protein was extracted from the cell samples with an RIPA buffer solution containing the protease inhibitor bicinchoninic acid (Solarbio, Beijing, China). The proteins were separated via polyacrylamide gel electrophoresis. Next, the proteins were transferred to a polyvinylidene fluoride membrane and blocked with 5% skim milk for 2 h. After sealing, the membranes were washed with TBST 3 times for 10 min each. The membrane was incubated with the primary antibody at 4 °C overnight and then washed with TBST 3 times for 10 min each. Then, the membrane was incubated with the secondary antibody at room temperature for 2 h. The luminous solution (A solution, B solution) was added at a 1:1 ratio in the dark. A pipette was used to obtain a suitable amount of luminous solution to cover the polyvinylidene fluoride film, which was exposed on the ECL luminous instrument; then, images were collected. The antibodies used in this study include MAP3K2 Rabbit mAb (MEKK2, R24953, ZENBIO, Chengdu, China), GAPDH Rabbit mAb (A19056, ABclonal, Wuhan, China), and HRP Goat Anti-Rabbit IgG (AS014, ABclonal, Wuhan, China). GAPDH was used as a loading control. Positive control (PC) is validated mouse embryonic stem cells used to verify the specificity of the MAP3K2 antibody.

Cells were lysed in ice-cold RIPA lysis buffer (R0020, Solarbio), and protein levels were quantified using a BCA protein assay kit (P0012S, Beyotime). The extracted cellular proteins were mixed with loading buffer (v/v = 4:1) (P1040, Solarbio) and boiled for 10 min. Protein samples (30 μg per well) were separated by 8% SDS–PAGE gels and transferred to PVDF membranes. The membranes were blocked at room temperature with 5% BSA in 1 × TBST (0.2% Tween-20, pH = 7.6) buffer for 2 h and then incubated with primary antibodies against GAPDH (1:100,000, A19056, ABclonal, Wuhan, China) and ARID5B (1:2000, NBP1-83622, Novus, Shanghai, China) at 4 °C overnight. After being washed with 1 × TBST buffer four times (10 min each time), the membranes were then incubated with HRP-conjugated mouse anti-rabbit (1:5000, AS061, ABclonal) and goat anti-mouse (1:5000, AS003, ABclonal) secondary antibodies at room temperature for 1 h. Protein bands were visualized by chemiluminescence using an ECL reagent (PE0010, Solarbio) and photographed by a Tanon-5200 Chemiluminescent Imaging System (Tanon, Shanghai, China). Image-Pro Plus (v 6.0) software was used to perform the densitometric semiquantitative analysis of protein bands. The protein expression was normalized against GAPDH.

Following deep anesthesia induced by sodium pentobarbital, the mice were sacrificed, and the hippocampus were obtained and pulverized in a lysis buffer. After centrifugation, the protein concentration of the liquid above was measured using the bicinchoninic acid kit provided by Boster, Ltd., Wuhan, China. Afterward, 20 μg of every nuclear protein sample underwent separation on 10% SDS‐PAGE gels and were then transferred onto a polyvinylidene fluoride membrane. Afterward, the membrane was obstructed using 5% BSA for a duration of 1 h at room temperature. Following this, it was incubated with specific primary antibodies overnight at a temperature of 4°C. Following three washes, the blots were exposed to the suitable secondary antibody for a duration of 1 h at ambient temperature. Immuno‐reactivity was quantified by densitometry using enhanced chemiluminescence.
The antibodies utilized in this study included anti‐GAPDH (A19056; ABclonal) at a dilution of 1:1000, anti‐ACSS2 (A12334; ABclonal) at a dilution of 1:1000, and anti‐HDAC2 (A19626; ABclonal) at a dilution of 1:1000.

Western blot analysis was performed as described previously 25 . Primary antibodies used were anti-WTX (A20434; Abclonal), anti-TGF-β2 (A3640; Abclonal), anti-phospho-SMAD3 (AP0727; Abclonal), anti-SMAD3 (A19115; Abclonal), anti-LC3B (A19665; Abclonal) and anti-GAPDH (A19056; Abclonal).