M00653

Protein samples were boiled in 1 × SDS loading buffer and separated using a 4–12% gradient Bis-Tris Gel (M00653; GenScript) by SDS-PAGE. Subsequently, the separated proteins were transferred onto a 0.45 µm PVDF membrane (IPVH00010; Millipore) and blocked with 5% skimmed milk at room temperature for 2 h. Then, the blots were incubated for another 2 h at room temperature with the following primary antibodies: anti-FLAG (1:1000, F1804; Sigma), anti-GFP (1:1000, K200047M; Solarbio), anti-cytochrome c (1:1000, MA5–11674; Invitrogen), anti-Drp1 (1:1000, ab184247; Abcam), anti-SipA (Shanghai Willget Biotech Co., Ltd.), anti-GroEL (1:1000, ab82592; Abcam), anti-Tomm20 (1:1000, ab56783; Abcam), and anti-β-Actin (1:1000, CW0096; CWBIO). To detect proteins, the blots were incubated with the corresponding horseradish peroxidase (HRP)-conjugated anti-rabbit (EF0002; SparkJade) or anti-mouse (EF0001; SparkJade) secondary antibodies at room temperature for 1 h. Finally, the blots were visualized with an ECL detection kit (D601039; Sangon Biotech) using an Amersham^TM Imager 600 system (General Electric). Immunoblotting bands were quantified using the ImageJ software. The data were collected from three biological replicates.

Neurons (1 × 10^5 cells/cm²) were lysed in RIPA buffer (Beyotime, China). Protein extract quantification was performed using a BCA protein assay kit (Beyotime, China). Denatured protein samples were separated with 4–12% (GenScript, M00653) sodium dodecyl sulfate-polyacrylamide and then transferred onto the polyvinylidene difluoride membranes. Membranes were blocked with skim milk solution for 2 h followed by primary antibodies incubation at room temperature for 2 h. Primary antibodies for NRXN1 (Santa Cruz, sc136001) were diluted 1:100 in PBS-Tween-20. Primary antibodies for GAPDH (10494, Proteintech) were diluted at 1:5000. The membranes were washed thrice (about 10 min/wash) using Tween-20 (pH 8.0) and Tris-buffered saline (TBST). Subsequently, membranes were incubated at room temperature for 2 h, using the horseradish-peroxidase-conjugated secondary antibodies (SA00001-1; SA00001-2, Proteintech, dilution WB: 1:5000). Membranes were then washed thrice with PBST, and the enhanced chemiluminescence substrate (Millipore, USA) was used to develop the blots. The protein bands were imaged in a gel image processing system. GAPDH was used as the protein loading control. Relative expression of the target protein was determined by the corresponding ratio of the optical density values of the internal reference and the target protein band using ImageJ.

Skeletal muscle tissues were lysed in RIPA buffer containing PMSF. The quantification of proteins was executed using a BCA (bicinchoninic acid) protein assay kit (PA115, TIANGEN, Beijing, China). Proteins were separated via SDS-PAGE using 4–12% precast gels (M00653, Genscript, Nanjing, China) followed by blotting onto nitrocellulose membranes. To block nonspecific binding, membranes were immersed in 5% non-fat milk solution in TBST (Tris-buffered saline containing 0.1% Tween-20) for 1 h. Primary antibodies used in the present study are shown in Table 1. Then, HRP-conjugated secondary antibodies (goat anti-rabbit or goat anti-mouse) were applied for 1h. Protein bands were visualized with the DAB chromogenic reagent kit (PA110, TIANGEN) and protein expression levels were analyzed using ImageJ.