Sc 514032

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.

Thirty hours after transfection, SKBR3 cells were lysed in RIPA buffer, and protein concentrations were measured by Bradford assay. Total protein was separated by SDS-PAGE using a 10% polyacrylamide gel and electroblotted onto a PVDF. The membrane was immunoblotted overnight at 4°C with primary antibodies against phospho-AKT (Santa Cruz, USA, sc-514032), AKT (cell signaling), and β-actin (Santa Cruz, USA, sc-130301) and a secondary goat anti-mouse antibody (BIORAD, USA, 1721011) were diluted according to the manufacturers' instructions and was incubated with the membrane for 1 h after three washes with TBST. Signals were detected with ECL detection reagent (Pierce, Rockford, IL). The images were obtained by Quantity One (Bio-Rad). Original blots are shown in Supplementary Figure 1.

Cells were fixed in 4% PFA in Pagano solution (250 mm sucrose, 25 mm MgCl₂, 2.5 mm KCl, 25 mm HEPES, pH 7.4; + phosphatase inhibitors for phospho immunostaining) for 15 min at room temperature, rinsed 3× for 5 min in Pagano, and incubated with blocking solution (Pagano + 0.25% Saponin + 2% BSA) for 1 h. Cells were incubated in primary antibodies diluted in blocking solution for 1 h, rinsed 3× for 5 min with Pagano solution, incubated with appropriate secondary for 1 h, then rinsed 3× for 5 min with Pagano solution. The primary antibodies used were anti-MAP2 (1:250, Chemicon, ab5756), anti-SMI-312 (1:1000, Covance, SMI312R), anti-p-Akt (1:100, Santa Cruz, sc-514032), Cat-315 (1:20, from R. Matthews; Dino et al., 2006 (link)), CS56 (1:200, Abcam, ab11570), and anti-DCX (1:500, gift from C. Walsh; Gleeson et al., 1999 (link)) polyclonal sera. Appropriate Alexa Fluor 488-conjugated, Alexa Fluor 555-conjugated, and Alexa Fluor 647-conjugated secondary antibodies were used. Hoechst 33342 (2 μg/ml; Invitrogen) was used to counterstain nuclei. Immunohistochemistry for explants was performed as previously described (Nichols et al., 2013 (link)). Images were collected with a Zeiss LSM780 laser scanning confocal microscope (SUNY Upstate Advanced Fluorescence Imaging Core).