Nb100 1607

Cells or minced sciatic nerves were mixed with RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) at 4 °C for 10 min or 30 min, respectively. The lysate was centrifuged at 13,200 rpm at 4 °C for 15 min and the supernatant was quantified using the BCA protein quantification kit (P0012, Beyotime). The 20–30 µg of protein were used for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). After blotting onto PVDF membranes (Millipore, Bedford, MA, USA), the membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated overnight at 4 °C with the primary antibodies as follow: Stat1 (14994, Cell Signaling, 1:1000), MAG (34-6200, Invitrogen, 1:100), Egr2 (NB110-59723, Novus, 1:500), Nab2 (PA5-75321, ThermoFisher, 1:500), P0 (NB100-1607, Novus, CO, USA; 1:500), Rab11fip1 (sc-517228, Santa Cruz, CA, USA; 1:500) and GAPDH (60004-1-Ig, Proteintech, Chicago, USA; 1:20,000). After 3 washes with TBS, the membrane was incubated with the corresponding HRP-conjugated secondary antibody for 1 h at room temperature before detection using the Pierce™ ECL Western Kit (ThermoFisher). The membranes were detected with a scanner (Bio-Rad Hercules, CA, USA) to obtain grayscale images, data analysis was processed using Image J.

Cells fixed in 4% paraformaldehyde (PFA) for 30 min at room temperature were used for immunocytochemistry (ICC). PFA-fixed tissue was embedded in optimal cutting temperature compound (OCT) and cryostat cut into tissue sections (12 µm) for IHC. The fixed cells or tissue sections were blocked in 5% donkey serum plus 0.1% Triton X-100 in PBS (PBS-T) for 1–2 h at room temperature, and then incubated overnight at 4 °C with the following primary antibodies: S100β (S2532, Sigma, 1:200), MAG (34–6200, Invitrogen, 1:100), Stat1 (14994, Cell Signaling, 1:200), beta-III tubulin (TuJ1, MMS-435P, Covance, 1:250), P0 (NB100-1607, Novus, 1:50). After 3 washes with PBS, the cells or tissue sections were incubated with corresponding fluorescence-conjugated secondary antibodies (1:1,000; Jackson Immunoresearch, PA, USA) for 1 h at room temperature. The images were captured using a fluorescence microscope (Leica).