Pvdf membrane

The total protein form kidney tissue was obtained using the ProteinExt® Mammalian Total Protein Extraction Kit (DE101, TransGen Biotech. Beijing, China).The Easy II Protein Quantitative Kit was used to determine the protein concentrations (DQ111, TransGen Biotech. Beijing, China). The renal proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Solarbio, Beijing, China). The membranes were incubated overnight at 4 °C with the following antibodies: β-Actin (13E5, CST, USA), GRP78 (C50B12, CST, USA), JNK (CST, USA), P-JNK (81E11, CST, USA), Caspase-12 (CST, USA), Bcl-2 (D17C4, CST, USA), Bax (D3R2M, CST, USA), CHOP (D46F1, CST, USA), and anti-DDIT3 (phosphor S30, abcam, England). Then, the membranes were washed with TBST and incubated with a secondary antibody blocking solution for 2 h at room temperature. Proteins were detected on a DNR Bio Imaging system by using the NcmECL Ultra method according to the manufacturer’s instructions (Ncmbio, Suzhou, China). The Gel Quant system was used to quantify the expression of target proteins.

Western blot analysis was performed as previously reported [50 (link)]. Protein extracts of the tissues and cells were obtained using ice-cold RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, protease inhibitor [Beyotime, Shanghai, China]) containing phenylmethanesulfonyl fluoride (PMSF, Beyotime). Protein extracts were boiled in 5× SDS-PAGE sample loading buffer (Beyotime) for 10 min, and protein concentration was determined using a BCA protein assay kit (Beyotime). Up to 15 µg of protein were separated in SDS-PAGE gels (Beyotime) by electrophoresis, and proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Solarbio, Beijing, China). After membrane blocking in 5% nonfat milk for 2–3 h, the membranes were incubated with the primary antibodies overnight at 4 °C and then washed three times for 30 min in TBST (Tris-buffered saline and Tween-20) before incubation with the secondary antibodies (ZB-2301; Beijing Zhongshan Golden Bridge Technology Co., Ltd., Beijing, China) conjugated with horseradish peroxidase for 2 h at room temperature. The membranes were washed three times with TBST and further quantified using chemiluminescence with a super enhancer ECL kit (Novland, Shanghai, China). The quantification of western blot bands was performed using ImageJ. All experiments were performed at least three time.

The soluble proteins were denatured by adding 4× SDS-PAGE Loading Buffer (TaKaRa) and heating at 95 °C for 10 min. Samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in SDS-containing polyacrylamide gels (12%) and transferred to polyvinylidene fluoride (PVDF) membranes (Solarbio). Membranes were then blocked for 60 min with PBS containing 0.1% Tween-20 and 5% skimmed milk powder. Anti-mouse DNMT1 mAb (Abcam), anti-human TET1 mAb (Santa Cruz) and anti-mouse β-actin mAb (Cloud-Clone) were used as the primary antibodies for the following Western blotting detection, whereas goat anti-mouse IgG-HRP (Cloud-Clone) was used as the secondary antibody. Grayscale values of each band were measured with an ImageJ software. Grayscale ratio equals to the value of target protein divided by the value of β-actin, which reflects the relative expression of target proteins shown under the corresponding bands.