Rabbit anti wnt5a

BV2 cells and mouse spinal cord L4‐5 segment tissues were digested in RIPA extraction buffer (Beyotime, China). Protein samples were separated by 10% SDS‐PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, United States) in tank transfer system (Bio‐Rad, United States). Membranes were blocked with 5% nonfat milk in buffer containing 0.1% Tween‐20 (TBST) for 1 h, washed three times in TBST, and incubated overnight at 4°C with primary antibodies including rabbit anti‐Wnt5a (1:1000 dilution; Proteintech; USA; 55,184‐1‐AP), rabbit anti‐CaMKII (1:1000 dilution; Proteintech; USA; 13,730‐1‐AP), rabbit anti‐NFAT (1:1000 dilution; Proteintech; US; 22,023‐1‐AP), rabbit anti‐GAPDH (1:10000 dilution; Proteintech; USA; 10,494‐1‐AP), rabbit anti‐iNOS (1:1000 dilution; Proteintech; US; 18,985‐1‐AP), and rabbit anti‐CD206(1:1000 dilution; Proteintech; US; 18,704‐1‐AP). After incubation with the HRP‐conjugated goat anti‐rabbit IgG secondary antibody (1:10000 dilution, Da‐UN, China), immunoreactive bands were detected by enhanced chemiluminescence (Millipore, United States). The protein bands were quantitatively analyzed using ImageJ software 1.8.0 (National Institutes of Health, Bethesda, MA, USA).

The cultured cells were collected and lysed in RIPA buffer (Beyotime) with protease inhibitors (Beyotime) after rinsing twice with PBS (precooled at 4°C). Protein was quantified with a BCA protein concentration determination kit (Beyotime), and then separated by SDS- polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene fluoride membranes. After blocking in 5% nonfat milk for 2 h at 25°C, the membranes were probed with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (Proteintech Group, Inc.). Primary antibodies used were rabbit anti-VEGF protein (1 : 1000, Proteintech Group, Inc.), rabbit anti-Wnt5a (1 : 1000, Proteintech Group, Inc.), rabbit anti-TGF-β1 (1 : 1000, Proteintech Group, Inc.) and rabbit anti-Tubulin (l : 1000, Proteintech Group, Inc.). We used BeyoECL Star (Beyotime) reagent to detect protein.