β tubulin

Cells were lysed in ice-cold RIPA cell buffer (P0013B, Beyotime Biotechnology, China) supplemented with protease inhibitor cocktail. The proteins (40–100 μg) were separated on 10–15% PAGE gels and electrotransferred onto polyvinylidene fluoride membranes. After blocking with 5% skimmed milk, the membrane was incubated with specific primary antibody and horseradish peroxidase-conjugated secondary antibody. The antibodies used are Flag (GNI4110-FG-S, GNI, Japan, 1:1000), HA (GNI4110-HA-S, GNI, Japan, 1:1000), Stat3 (12640S, Cell Signaling Technology, USA,1:1000), phospho-Stat3^Y705 (9131S, Cell Signaling Technology, USA, 1:1000), Socs3 (sc-73045, Santa Cruz, 1:1000), Prdm14(D221722, BBI, China, 1:1000), H3K27me3 (39055, Active Motif, China, 1:1000), H3 (4620s,Cell Signaling Technology, USA, 1:1000) and β-Tubulin (200608, ZENBIO, China, 1:2000). The band density was analyzed with ImageJ according to ImageJ User Guide. Briefly, we inverted the greyscale images and select sample bands with rectangular selections. The proteins levels were normalized with respect to the β-Tubulin level, and the grayscale ratio of protein/β-Tubulin was calculated and visualized with GraphPad Prism 8.0.

Western blot (WB)analysis was performed as previously described [20 ]. Briefly, brains were removed immediately after anesthetization and decapitation were performed. Samples were collected from the object region of brains and lysed in RIPA buffer containing protease and phosphatase inhibitors (Sigma). Then protein extract was obtained after centrifugation. Equal amounts of protein (40 mg) were loaded onto each lane of SDS-PAGE gels. After gel electrophoresis, proteins were transferred onto nitrocellulose membranes (Millipore) and blocked in nonfat milk at room temperature for 2 h. Then membranes were incubated in primary antibodies at 4 °C for 24 h and incubated in appropriate secondary antibodies at room temperature for 1 h subsequently. The following antibodies were used: Iba-1(Wako), iron regulatory protein 1 (IRP1, ZenBioScience), divalent metal transporter 1 (DMT1, MyBioSource), transferrin receptor1 (TfR1, ZenBioScience), ferritin (MyBioSource), ferroportin1 (Fpn1, MyBioSource), β-actin (ZenBioScience) and β-tubulin (ZenBioScience). Protein bands were visualized using nickel-intensified DAB solution, and band densitometry analysises were performed using Image J software (National Institutes of Health). The housekeeping proteins β-actin or β-tubulin were used as a loading control.

Protein isolation was performed from the hepatocyte using protein extraction kit (BestBio Biotech Co. Ltd., Shanghai, China), and the BCA protein assay kit (BestBio) was used to determine the concentration of protein samples. Western blotting was performed with following the method described previously (Cui et al., 2020b ) with the following primary antibodies: anti-PPARα (1:2,000, ab233078, Abcam, Cambridge, UK), anti-PCNA (1:1,000, A0264, ABclonal Technology, Wuhan, China), anti-CDK2 (1:500, 120395, Zen-Bio, Chengdu, China), anti-caspase-3 (1:5,000, ab214430, Abcam) and caspase-8 (1:1,000, ab227430, Abcam), and caspase-9 (1:1,000, bs-20773R, Bioss, Beijing, China), anti-PDK1 [1:1,000, #3062, Cell Signaling Technology (CST), Boston, United States], anti-p-PDK1 (1:1,000, #3061, CST), anti-AKT (1:1,000, #9272, CST) and p-AKT (1:1,000, #9271, CST), and anti-Gsk3-β (1:1,000, A2081, ABclonal) and p-Gsk3-β (1:1,000, AP0261, ABclonal). Secondary antibodies: goat anti-mouse (1:5,000, 511103, Zen-Bio) and goat anti-rabbit (1:5,000, 511203, Zen-Bio). β-Tubulin (1:1,000, 250063, Zen-Bio) was used as a reference.