Ab23393

All samples were resolved on a 7% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in 4% milk in TBS-T (TBS, pH 7.4, and 0.1% Tween 20) for 1 h at room temperature. Primary antibodies [α-tubulin (1:30,000, T9026, Sigma-Aldrich), actin (1:1000, SC-1616-G, Santa Cruz Biotechnologies), Npn1 (1:1000, AF566, R&D Systems), or Sema3A (1:1000, ab23393, Abcam)] were diluted in 3% bovine serum albumin (BSA) and incubated overnight at 4°C. The following day, membranes were washed and incubated with appropriate horseradish peroxidase (HRP)-linked secondary antibodies (1:10,000) in 3% BSA (Jackson ImmunoResearch) followed by visualization with a chemiluminescent substrate (Thermo Fisher Scientific).

The cells were inoculated in the petri dish at the cell density of 1 × 10⁵/mL for cell crawling, and the cells were divided into scramble-transfected cells, mimic-transfected cells, and scramble-transfected cells treated with 20 ng/mL PDGF and mimic-transfected cells treated with 20 ng/mL PDGF. After the treatment, the cells were centrifuged at 1000 × g at 4°C for 5 min, and immunofluorescence was performed for identifying the fluorescent of the cells. Briefly, the cell smear was washed by PBS for three times and then fixed on ice acetone (01000356-25g, Beijing Ouhe Technology Co., Ltd., http://www.ouhechem.com/, China) for 15 min. Next, while the cell smear was dried, PBS was used to wash the cells for three times. TCF21/Pod1 antibody (C07617Cy3, Signalway Antibody, USA) and MTAP antibody (ab23393, 1 : 100, Abcam, USA) were added into the cells, respectively, and incubated together at 4°C in a heat preservation box incubation. Next, conjugated secondary antibody was added into the cells at 37°C for 2 h, and the cells were washed by PBS for three times and then incubated with DAPI for 3 min. Finally, fluorescence of the cells was determined under a fluorescence microscope (Delta Optical IB-100, Delta Optical, Poland).

Whole-cell protein was isolated from ASCs using the Total Extraction Sample Kit (Sigma) according to the instruction. For western blot analysis, equal proteins were loaded on 12% SDS-PAGE gels and then separated completely using vertical electrophoresis at 120 V. After transferring onto a 0.5 μm pore size nitrocellulose membrane at 200 mA, the separated proteins were blocked with 5% bovine serum albumin and incubated with rabbit polyclonal anti-Sema3A(Abcam, ab23393) and mouse polyclonal anti-GAPDH primary antibodies diluted in 5% TBST for 8 hours at 4°C. The protein bands were visualized using the Odyssey Infrared Imager system (LI-COR) after incubation with corresponding fluorescent secondary antibody for 1 hour at room temperature.

Muscle and sciatic nerve (SN) tissues of both sexes were excised and homogenized in lysis buffer containing PBS, 1% Triton X-100 (Sigma-Aldrich), and 1× protease inhibitors (Roche Diagnostics), followed by centrifugation and collection of the supernatant. Protein concentration was determined using the Bio-Rad Protein Assay. Protein samples were denatured by boiling in SDS sample buffer, which were then electrophoresed in 10% polyacrylamide gels (SDS-PAGE). Proteins were transferred to a nitrocellulose membrane and then immunoblotted with appropriate primary antibodies: Sema3A (Abcam, ab23393; 1:1000); NRP1 (Abcam, ab81321; 1:1000); Sema3B (Abcam, ab48197; 1:2000); NRP2 (Cell Signaling D39A5, 1: 1000); GFP (Abcam ab13970; 1:5000); tubulin (1:10,000) and ERK (1:10,000), diluted in 5% (w/v) skim milk (BD Difco) in TBS-T, followed by species-specific HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories; 1:10,000) and visualized using a myECL imager (Thermo Fisher Scientific), according to the manufacturer's instructions. Quantification was performed using ImageJ software.