Gtx17441

The antibodies for NP, GAPDH, CD63, CD81, TSG101 and HSP70 were purchased from Genetex (no. GTX629633, GTX100118, GTX17441, GTX31831, GTX10255 and GTX11573). The antibodies for HA and calnexin were purchased from Millipore (no. 04–902 and AB2301). Cell lysates were prepared using M-PER mammalian protein extraction reagent (Thermo Scientific) with additional protease inhibitors, subjected to electrophoresis on a SDS-PAGE, and transferred onto a Hybond-P membrane. The membrane was probed with the indicated primary and appropriate secondary antibodies, detected using an enhanced chemiluminescence detection kit, and then imaged by Image-Quant LAS4000.

Total protein was extracted from EV using RIPA buffer (Nacalai, Kyoto, Japan) and the concentration was measured by a Bradford assay (Bio-Rad). An amount of 50μg of extracted protein was separated by electrophoresis in 7.5% SDS-polyacrylamide gels. The proteins were transferred to a polyvinylidene difluoride membrane (Millipore). After that membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for an hour at room temperature. Membranes were then incubated with primary antibodies, including rabbit anti-CD63 (GTX17441; GeneTex), rabbit anti-TSG101 (GTX118736; GeneTex), and rabbit apolipoprotein A1 (APOA1, GTX40453; GeneTex) at 1:1000 dilution overnight at 4°C. Membranes were washed with TBST, then incubated with HRP-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific) at 1:10,000 dilution. The positive signals were analyzed by a luminescence imager (Image Quant LAS4000; GE Health Care, Little Chalfont, United Kingdom) using chemiluminescence reagents (Merck Millipore).

Injured cord segments around the lesion epicenter with 0.5 cm length were obtained to detect the expression of NF200, while cells were harvested at the designed time points. Total protein was extracted with ice‐cold radioimmunoprecipitation assay (Beyotime Biotechnology) buffer containing 1% phenylmethylsulfonyl fluoride (Beyotime Biotechnology). Proteins were separated on 10% sodium dodecyl sulfate–polyacrylamide gel and electro‐transferred to polyvinylidene fluoride (Milipore) membrane. The membranes were incubated with primary antibodies against CD9 (ab 92726, 1:1500; Abcam), CD63 (gtx17441, 1:1000; Genetex), p‐NF‐κB (3033t, 1:2000; Cell Signaling Technology), p‐c‐Jun (3270s, 1:2000; Cell Signaling Technology), NF200 (1:500, ab134306; Abcam), β‐actin (A5441, 1:3000; Sigma), GAPDH (10494, 1:4000; Proteintech) at 4°C overnight. After washed with Tris‐buffered saline Tween 20, the membranes were incubated with appropriate horseradish peroxidase‐conjugated secondary antibodies at RT for 1 h. The bands were detected by enhanced chemiluminescence (Milipore) and visualized using a Bio‐Rad Image Lab system.