Polyclonal rabbit anti vav1

The spinal cord segments were harvested, postfixed, and sectioned. The sections were allowed to incubate with polyclonal rabbit anti-CD74 (1:100; Bioss), monoclonal mouse anti-GFAP antibody (1:500; Millipore Sigma), or polyclonal rabbit anti-Vav1 (1:100; Proteintech Group Inc) at 4 °C for 36 h. The sections were further reacted with the Cy3-labeled secondary antibody goat anti-rabbit IgG (1:400; Thermo Fisher Scientific) or the FITC-labeled secondary antibody donkey anti-mouse IgG (1:400; Thermo Fisher Scientific) at 4 °C overnight, followed by observation under a confocal laser scanning microscope (Leica).

Protein was extracted from cells with a buffer containing 1% SDS, 100 mM Tris-HCl, 1 mM PMSF, and 0.1 mM β-mercaptoethanol. Alternatively, protein was extracted from 0.5 cm cord segments after gecko tail amputation at 1 day, 3 days, 1 week, and 2 weeks, respectively. After centrifugation at 13,000 r/min for 30 min at 4 °C, 20 μg of total protein of each sample was loaded into a 10% SDS-PAGE gel and transferred to PVDF membranes (Millipore Sigma). The membrane was then blocked with 5% nonfat dry milk in TBS containing 0.05% Tween-20 (TBS-T) for 1 h and incubated with polyclonal rabbit anti-gMIF (1:50; GenScript), polyclonal rabbit anti-Vav1 (1:1000; Proteintech Group Inc), or polyclonal rabbit anti-CD74 (1:1000; Biorbyte) primary antibodies. After reaction with the second antibody conjugated with goat anti-rabbit or goat anti-mouse HRP (1:5000; Santa Cruz Biotechnology) at room temperature for 2 h, the HRP activity was detected using enhanced chemiluminescence. The membrane was scanned with a ChemiDOC XRS+ Imager (Bio-Rad). The data were analyzed using PDQuest 7.2.0 software (Bio-Rad). β-actin (1:5000; Cell Signaling Technology) was used as an internal control.