Senp1

After 48 h of transfection, the cells were lysed in RIPA buffer (Beyotime) supplemented with protease inhibitors cocktail (MCE, New Jersey, USA). Then the protein was separated by 10% SDS‐PAGE and transferred to a PVDF membrane (Millipore, Burlington, USA). After being blocked in 5% skimmed milk powder for 2 h, the membranes were incubated with primary antibodies against SENP1 (1:1000, Proteintech, Chicago, USA), β‐actin (1:5000, ABclonal, Boston, USA) or GAPDH (1:5000, ABclonal) overnight at 4℃. The protein band intensity was detected using ChemiDOCTMXRS⁺ imaging system (BIO‐RAD, Minnesota, USA).

The slides of tissue microarray were incubated with the primary antibody SENP1 (1:100, Proteintech) overnight at 4℃. IHC was performed as we described previously.⁸

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.