Ab169761

Electrophoresis gel, electrophoresis solution, electrophoresis solution, and tris-buffered saline with 0.1% Tween (TBST) were prepared prior. BCA was used to determine protein concentration, and each well was loaded equally. The gels were transferred to a 0.45-µm PVDF membrane (A10178785, GE, USA); 5% nonfat milk in TBST was used to block the membrane for 1 h at room temperature. The membranes were then incubated at 4°C overnight with primary antibodies for 16 h. The primary antibodies were as follows: anti-RGMa (1:10,000, ab169761, Abcam, USA); anti-BMP2 (1:1,000, YT5651, Immunoway, USA); anti-BMPR II (1:500, 220550, ZEN Bio, Chengdu); anti-YAP (1:1,000, 14074, Cell Signaling Technology, USA); anti-ZO-1(1:1,000, 40-2200, ThermoFisher Scientific, USA); anti-GAPDH (1:10,000, 20494-1-AP, Proteintech, Wuhan); and anti-beta Tubulin (1:4,000; 10068-1-AP, Proteintech, Wuhan). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG, SA-00001-2, Proteintech; goat anti-mouse IgG, SA00001-1, Proteintech) for 1 h at room temperature. Images were captured by the Fusion FX5 image analysis system (Vilber Lourmat, F77601 Marne-la-Vallée cedex 3, France).

Tissues from the gray matter at the spinal lesion sites were collected and lysed in radio-immunoprecipitation assay lysis buffer (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China), and the protein concentration was determined using a bicinchoninic acid kit (70-PQ0012, MultiSciences, Zhejiang, China). Then, the protein was separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% BSA for 1 h and then incubated with the primary antibodies to RGMA (1:10000, ab169761, Abcam), NF-200 (1:1000, #55453, CST), synaptophysin (1:1000, ab14692, Abcam), STAT3 (1:1000, ab68153, Abcam), p-STAT3 (1:1000, ab32143, Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:2000, ab9485, Abcam) at 4 °C overnight. Next, the membranes were incubated with the horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (1:2000, ab205719, Abcam) at room temperature for 1 h. The western blots were developed using an enhanced chemiluminescence kit (BB-3501, Amersham Pharmacia Biotech, Piscataway, NJ, USA) exposed by a Gel imager, captured by a Bio-Rad image analysis system (Bio-Rad, Inc., Hercules, CA, USA), and analyzed using the Quantity One v4.6.2 software.