Ab19031

Western blot analysis was performed as previously described.^{21 (link)} Protein concentration was measured by Enhanced Bicinchoninic Acid Assay
Protein Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China).
Protein samples (20 μg/lane) were loaded on a 12% sodium dodecyl sulfate
polyacrylamide gel electrophoresis to separate and electrophoretically
transferred to a polyvinylidene difluoride membrane (Millipore Corporation,
Billerica, MA, USA). The membrane was blocked with 5% bovine serum albumin (BSA;
BIOSHARP, Hefei, AH, China) for 1 h at room temperature. The membrane was
further incubated overnight at 4 °C with anti-Marcks antibody (ab217695; Abcam
Technology), anti-PKC antibody (ab19031; Abcam Technology), anti-MMP-9 antibody
(ab38898; Abcam Technology), and anti-β-actin antibody (ab8226; Abcam
Technology). Then, the membrane was incubated with corresponding horseradish
peroxidase-conjugated secondary antibodies (ZSGB Biotech Co., Ltd., Beijing,
China) for 2 h at room temperature. The signal was developed using an enhanced
chemiluminescence (ECL) kit (Beyotime Institute of Biotechnology, Shanghai,
China) and then exposed to X-ray films. The films were scanned using an Epson
Perfection 2480 scanner (Seiko Corp, Nagano, Japan). The relative quantity of
protein was analyzed based on densitometry analysis by use of the Image J
program and normalized to that of loading controls.

Initially, total protein in the sciatic nerve lysate supernatant was estimated by BCA protein assay kit (BCA-1, Sigma, USA). Aliquots of the lysates containing 40 μg of protein were subjected to denatured SDS-PAGE on polyacrylamide gels (MiniProtean TGX precast gels, BioRad). After transferring onto nitrocellulose membrane, the protein bands were blocked with 10 ml blocking buffer containing 5% non-fat dried milk in TBST (25 mM Tris-HCl, 137 mM NaCl, 2.65 mM KCl, and 0.05% Tween 20; pH 7.4) for 2 h at room temperature. The blots were washed in TBST (x5) and probed with aldose reductase (abcam; ab175394) and protein kinase C (abcam; ab19031) primary antibodies (1:500 dilution block buffer). Beta actin monoclonal antibody (RM112) was used for blot normalization (loading control). After incubating overnight at 4 °C with shaking, anti-rabbit HRP-conjugated secondary antibody (1:2000 dilution) was added and incubated with rocking for 1 h at room temperature. The antibody-reactive bands were visualized by an enhanced chemiluminescence (ECL) detection kit (Pierce, Thermo Scientific).