Sumo2 3

Total proteins were extracted from cells or tissues using the M-PER mammalian protein extraction reagent (Thermo Pierce, Rockford, USA) and measured using the BCA Protein Assay (Thermo Pierce). Protein samples (50 μg per assay) were separated on SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% skim milk and then incubated overnight at 4 °C in the presence of primary antibodies. The primary antibodies for p21, p53, KHSRP, c-Myc, EPCAM, and β-actin were purchased from Cell Signaling Technologies (CST, Danvers, MA, USA), p-c-Myc antibody was from Santa Cruz (CA, USA), and IκBα, Rb1, STAT3, histone-H3, SUMO1, and SUMO2/3 antibodies were from the Proteintech Company (Chicago, IL, USA). The membranes were washed 3 times in Tris-buffered saline with Tween-20 and incubated with horseradish peroxidase-conjugated secondary antibody (CST, MA, USA) for 1 h at room temperature (RT). After washing 3 times with 15 mL of TBST, Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, USA) was added, and the bands were imaged with the Chemidoc XRS⁺ electrophoretic imaging system (Bio-Rad, Hercules, CA, USA). Density scanning of each protein band was performed using Image Lab software (Bio-Rad, Hercules, CA, USA).

BCA Protein Colorimetric Assay Kit (Elabscience Biotechnology Co., Ltd., China) was used to quantify the total protein of brain cortex tissues and SY5Y cells. Samples with an equal amount of protein were loaded and separated via sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The proteins were transferred to the appropriate polyvinylidene fluoride membrane (Millipore, USA), blocked with 5% skimmed milk for 1 h, and thens incubated with diluted antibodies at 4°C overnight. For the in vivo experiments, Drp1 (1:2000), Fis1 (1:1500), MFF (1:8000), OPA1 (1:2000), Mfn1 (1:1000), Mfn2 (1:2000), and β‐actin (1:5000) antibodies were purchased from Proteintech Group, Inc., China. Meanwhile, for the in vitro experiments, the antibodies included Drp1 (1:800; AiFang Biological, China), Fis1 (1:1500; Bioss, China), OPA1 (1:1000; AiFang Biological, China), Mfn1 (1:1000; Affinity, China), Mfn2 (1:500; AiFang Biological, China), and SENP6 (1:1000; Abcam, USA). Furthermore, MFF (1:2000), SUMO1 (1:2000), SUMO2/3 (1:500), SENP1 (1:2000), SENP2 (1:1000), SENP3 (1:800), SENP5 (1:3000), and β‐actin (1:5000) were obtained from Proteintech Group, Inc., China. Then, the membranes were washed and incubated with secondary antibodies for 2 h at room temperature. After rewashing with TBST buffer, the membranes were added ECL reagent to enable the detection of the protein bands.