Carmassi bradford reagents

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.

The contusion epicenter of sciatic nerve tissue (2 cm) was lysed in RIPA buffer containing phenylmethanesulfonyl fluoride (the volume ratio is 100:1) for 30 min and clarified by centrifugation at 12,000 rpm for 15 min. Then, the extracts were quantified with Carmassi Bradford reagents (Thermo, Rockford, IL, USA). 80 μg protein was separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto PVDF membranes (Millipore, USA). After blocking with 5% skim milk for 1.5 h, membranes were probed with primary antibody (GRP-78, ATF-6 and Cleaved-caspase 12, 1:1000, Abcam; XBP-1 and CHOP 1:300, Santa Cruz; GAPDH, 1:10000, Bioworld). Subsequently, blots were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (1:10000) [50 (link), 51 (link)]. Finally, the immunoreactive bands were scanned via optical density measurements using the ChemiDoc XRS+ Imaging System (Bio-Rad). Each sample was blotted 3 independent times, and the densitometric values of the bands were quantified with the Image Lab software (Bio-Rad).