Senp5

Lysate, IP or His‐PD Samples were resolved by SDS–PAGE (10–15% gels) and transferred to Immobilon‐P membranes (Millipore Inc.), which were then immunoblotted with the following antibodies against: β‐actin (Sigma), CHIP (Cell Signaling), Drp1 (Cell Signaling), Fis1 (Proteintech), Flag (Proteintech), FTH1 (Cell Signaling), GAPDH (Santa Cruz biotechnology), GST (GE Healthcare), LC3 (Cell Signaling), p62 (Cell Signaling), SENP3 (Cell Signaling), SENP5 (Proteintech), SUMO‐1 (Santa Cruz biotechnology), SUMO‐2/3 (Cell Signaling; MBL) or Tom20 (Santa Cruz biotechnology). Immune complexes were detected using either HRP‐conjugated secondary antibodies (Sigma) followed by enhanced chemiluminescence (GE Healthcare) or using fluorescent secondary antibodies (LI‐COR).
Each immunoblot presented is representative of at least three experiments carried out using different cell populations.

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.

Immunohistochemistry was performed as described. Briefly, 5-micron thick formalin-fixed paraffin embedded human tissue sections were stained with the SENP5 (#19529-1-AP, Proteintech, USA), Ki67 (GB111499, Servicebio, China), γ-H2AX(GB111841, Servicebio, China), RAD51 (#ab1837, abcam, US) or p-CHK1 (#ab47318, abcam, US) antibody per manufacturer’s instructions. TUNEL staining was conducted to evaluate apoptosis in resected tissue according to the manufacturer’s instructions (Servicebio, Wuhan, China). Stained slides were digitized using the panoramic slice scanner (3DHISTECH, Hungary) with a 40× objective. Three fields of view per section were used to determine the mean and standard error of the mean of positively staining cells.