Sc 81434

Total protein from the spinal cord tissue was purified using protein extraction reagents containing 1% protease and phosphatase inhibitors. The protein concentration of each sample was quantified with Carmassi Bradford reagents (Thermo). An equivalent amount of protein (60 µg) was separated by 10% SDS‐PAGE and transferred onto PVDF membranes (Bio‐Rad). After blocking with 5% (w/v) non‐fat milk, the membranes were further incubated with primary antibody solutions overnight at 4°C. The following primary antibodies were including: TrkA (ab‐76291, Abcam, 1:5000), FGFR1 (ab‐58516, Abcam, 1:1000), P‐AKT (sc‐514032, Santa Cruz, 1:1000), AKT (sc‐81434, Santa Cruz, 1:1000), P‐ERK (sc‐16982, Santa Cruz, 1:1000), ERK (sc‐514302, Santa Cruz, 1:1000), Bcl‐2 (60178‐1‐Ig, proteintech, 1:3000), Bax (60267‐1‐Ig, proteintech, 1:2000), cleaved caspase‐3 (sc‐373730, Santa Cruz, 1:500), GAP43 (ab75810, Abcam, 1:10 000), GFAP (ab7260, Abcam, 1:2000), NF‐200 (ab8135, Abcam, 1:5000) and GAPDH (K200057M, Solarbio, 1:5000). After three washed with TBST, the membranes were incubated with a 1:10 000 dilution of horseradish peroxidase‐conjugated secondary antibodies for 60 minutes at room temperature. Finally, signals were visualized by Chemi DocXRS + Imaging System (Bio‐Rad). All experiments were repeated three times.

BCA protein assay was used to measure protein concentration. Western blot procedure was conducted as previously [31 (link)]. Equivalent protein amount (40 μg) was loaded and separated by 8–12% SDS–polyacrylamide gels and then transferred onto Immobilon-P Membranes (PVDF, IPVH00010, ISEQ00010, Millipore). The membranes were incubated at 4 °C overnight with the indicated primary antibodies. After washing three times with PBS containing 10% Tween-20 (PBST), the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies. SuperSignal™ West Pico PLUS (#34577, Thermo Scientific) was used for visualization. Capillary electrophoresis experiment was performed on WES-Automated Western Blots with Simple Western (Proteinsimple, Bio-techne) following guidelines of sampler kits (12–230 kDa separation module, #SM-W001,). Antibodies against NGB, VEGFR2 (sc-6251), AKT (sc-81434) and β-actin (sc-8432) were purchased from Santa Cruz Biotechnology. Antibodies against p-VEGFR2 (#2478), pAKT (#4060), p-Src (#6943) and p-ERK 1/2 (#4370) were purchased from CST. GPR35 (DF4973, Affinity), E-cadherin (abcam, ab76055), Vimentin (abcam, ab8069), N-cadherin (BD, 2248858). All assays were performed three times independently.