Sc 16982

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.

EL4 cells transfected overnight with pEGFP-C3-siOCILRP2 were stimulated with anti-CD3/CD28 antibodies. For the other two groups, EL4 cells were stimulated with or without anti-CD3/CD28 antibodies. Cell lysates were prepared in SDS sample buffer (Tris-HCl, SDS, and 20% glycerol) and detected using SDS-PAGE. The proteins were electrotransferred onto Immobilon-P PVDF membranes (0.45 µm, Millipore, USA) and probed with the appropriate primary antibodies against the following: p-Raf-1 (Ser 338) (sc-12358, Santa Cruz, 1∶200), caspase-8 p18 (G-1) (sc-166596, Santa Cruz, 1∶500), caspase-3 p17 (B-4) (sc-271028, Santa Cruz, 1∶500), JNK1 (D-6) (sc-137018, Santa Cruz, 1∶500), p-JNK1/2/3 (T183+Y185) pAb (BS4322, Bioworld Technology, 1∶500), ERK 1/2 (H-72) (sc-292838, Santa Cruz, 1∶200), p-ERK 1/2 (Thr202/Tyr204) (sc-16982, Santa Cruz, 1∶200), IκB-alpha (N-terminus) mAb (MB0106, Bioworld Technology, 1∶1000), β-actin (Sigma, 1∶5,000), and β-tubulin (Sigma, 1∶5000)). After incubation with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz, 1∶10,000) for 3-4 h, the membranes were washed with PBST. Immunoreactivity was visualized using an ECL system (Perkin Elmer, USA), and densitometry scanning of the intensity of the bands was quantified using ImageJ.