Purified β-actin, β-tubulin and cytoskeletonal protein aggregation kits were purchased from Cytoskeleton, Inc. (Denver, CO, USA). Dulbecco's Modified Eagle's medium (DMEM) and Lipofectamine 2000 reagent were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Anti-β-actin monoclonal antibody was commercially available from Genetex, Inc. (cat. no. GTX109639; Irvine, CA, USA). Anti-β-tubulin antibody (cat. no. T4026) and phenylmethylsulfonyl fluoride (PMSF) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The peroxidase-conjugated affinity goat anti-mouse IgG secondary antibody was obtained from Roche Diagnostics (Basel, Switzerland). An expression vector pCMV-HA (cat. no. Trans 18–35; Clontech; Takara Bio Inc., Mountain View, CA, USA) encoding for the full-length β5 subunit cDNA used for transfection was constructed by SyngenTech Co., Ltd. (Shanghai, China). The enhanced chemiluminescence (ECL) assay kit (cat. no. PA112) used in western blotting was commercially available from Tiangen Biotech Co., Ltd. (Shanghai, China).

PC12 cells (American Type Culture Collection, Rockville, MD, USA) were routinely cultured in DMEM supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) in a 37°C incubator with an atmosphere of 5% CO₂. PC12 cells were subjected to oxidative stress by treatment with various concentrations of H₂O₂ (100, 200 and 300 _{µM) for 48 h, or with 300} µM H₂O_{2 for different time durations (24 or 48 h).} PC12 cells in the control group were treated with culture medium only.

GSH is one of the most important factors protecting from oxidative attacks by reactive oxygen species, since it functions as a reducing agent and free-radical trapper (14). In addition, TBARS is a marker of lipid peroxidation and oxidative damage (15). Therefore, the present study investigated the levels of GSH and TBARS in PC12 cells that were subjected to H₂O₂-induced oxidative stress in order to determine the effect of oxidative stress in these cells.

For TBARS detection, 100 µl plasma were incubated with 500 µl Tris-HCl (Amresco Inc., Solon, OH, USA) and 500 µl 35% trichloroacetic acid (TCA; Merck KGaA, Darmstadt, Germany) for 10 min at room temperature. Next, the samples were incubated with 2 M Na₂SO₄ and 55 mM TBA solution at 95°C for 45 min, and then cooled on ice for 5 min. Subsequent to treatment with 70% TCA, the samples were centrifuged at 12,000 × g for 3 min. The TBARS product was measured at 530 nm using a spectrophotometer (Ultrospec 500 Pro; GE Healthcare Life Sciences, Chicago, IL, USA).

For the detection of GSH, 5% TCA, 67 mM sodium/potassium phosphate buffer and 1 mM 5,5′-dithiobis(2-nitrobenzoic acid) (Vicmed, Xuzhou, China) were mixed with 20 µl erythrocyte lysate and incubated in the dark at room temperature for 45 min. GSH production was determined at 412 nm using a spectrophotometer (Shimadzu-1640; Shimadzu Corp., Kyoto, Japan).

PC12 cells were washed with phosphate-buffered saline (PBS) (3×10⁶ cells/ml) and lysis treated a lysis buffer containing 50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, phosphatase inhibitors (100 mM Na3VO4 and 10 mM NaF) and a protease inhibitor (1 mM PMSF). The cell homogenate was incubated with KCl at a final concentration of 500 mM in the presence of 0.5% v/v Triton X-100 (Sigma-Aldrich, Munich, Germany). After 2 h of incubation, the supernatant (namely the monomer) and the pellet (namely the polymer) were separated by centrifugation at 16,000 × g for 20 min at 4°C. Subsequently, the same amounts (50 _µg) of supernatant and pellet were loaded onto separate lanes and separated by 10% SDS-PAGE. The samples were then probed with antibodies against β-tubulin and β-actin. The integrated densities of the protein bands were obtained using ImageJ software (version 1.3; National Institute of Mental Health, Bethesda, MD, USA) to calculate the ratio of monomer to polymer in β-tubulin and β-actin under KCl conditions.

AG is a nucleophilic hydrazine and nontoxic small molecule, which exerts pharmacological effects indicating that it has antioxidant properties (16). The protective effects of AG have been investigated in several experimental animal models, including oxidative stress-induced lung and liver injuries (17), and ischemia/reperfusion injury (18). AG appears to have useful properties that may block the protein carbonylation in PC12 cell lines exposed to H₂O₂; therefore, AG pretreatment was conducted prior to H₂O₂ exposure in the present study. PC12 cells in the experimental group were treated with 0.5 mM AG (AB120123; Abcam, Cambridge, MA, USA) for 30 min, while PC12 cells in the control group were treated with culture medium only. After 300 µM H₂O₂ treatment for 48 h, the changes in the carbonylation level were determined using western blot analysis.

Prior to H₂O₂ treatment, cells were suspended at 5×10⁵ cells/ml in DMEM supplemented with 10% fetal bovine serum and transfected with an empty vector or the β5 plasmid using the Lipofectamine 2000 reagent, according to the protocol provided by the manufacturer. At 2 days after transfection, the expression of β5 in the transfected cells was examined by western blot analysis.

A pull-down/western blot method was used to determine the extent of protein carbonylation. Briefly, biotinylation of protein carbonyls was performed through reaction of cells with biotin hydrazide (Bioworld Technology, Inc., St. Louis Park, MN, USA) in the presence of sodium cyanoborohydride (Santa Cruz Biotechnology, Santa Cruz, CA, USA). An aliquot (200 µg) of these protein homogenates was kept for western blot analysis, while streptavidin-agarose (Thermo Fisher Scientific Inc.) was added to the remainder homogenate in order to isolate the biotinylated proteins. Next, SDS sample buffer was used to elute the proteins from the avidin agarose beads, followed by western blot analysis.

At 2 days after transfection, PC12 cells were washed with phosphate-buffered saline (PBS) and treated with a lysis buffer containing 50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, phosphatase inhibitors (100 mM Na₃VO₄ and 10 mM NaF) and a protease inhibitor (1 mM PMSF). The lysates were centrifuged at 11,000 × g for 10 min at 4°C, and the supernatant was collected. The protein concentrations were then quantitated using the Lowry protein assay method. An equal amount of sample (50 µg) was subjected to 10% SDS-PAGE and was blotted onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Subsequently, the samples were blocked in PBS-Tween 20 (PBST) with 5% nonfat dry milk, and the membranes were incubated with primary antibodies against β-tubulin (1:1,000) and β-actin (1:5,000) at appropriate dilutions in PBST overnight at 4°C. The membranes were then washed three times with PBST solution, followed by incubation with goat anti-mouse secondary antibody (1:3,000) in PBST. Subsequently, the proteins were probed with horseradish peroxidase (HRP)-phytohemagglutinin-L and HRP-concanavalin A lectin. Visualization of the results was performed by fluorography using an ECL assay system.

Densitometric analysis of the western blot results was performed by the ImageQuant TL control center (GE Healthcare Life Sciences). Statistical analysis was performed using GraphPad Prism version 5 (GraphPad Software, Inc., La Jolla, CA, USA). One-way analysis of variance was used to compare the parameters measured in the different study groups.

Upon treatment of PC12 cells with increasing concentrations of H₂O₂, the level of GSH was reduced compared with that in the untreated control group (Fig. 1A). The GSH content in PC12 cells was reduced to 40% compared with the untreated control, and after treatment with GSH for 24 or 48 h, the GSH content decreased to ~50 and 30%, respectively, which indicated a clear time and dose-dependent effect. By contrast, the level of TBARS increased steadily with increasing concentrations of H₂O₂, and was ~3-fold higher than the control group after treatment with 300 µM H₂O₂ for 48 h (Fig. 1B). These results suggested that H₂O₂ induced oxidative stress.

Subsequently, the β-actin and β-tubulin carbonylation was investigated. A significant increase (P<0.05) was observed in the proportion of carbonylated β-actin and β-tubulin (Fig. 1C and D) in H₂O₂-treated cells compared with the untreated control cells, in a dose-dependent manner. Therefore, these results indicated that the increase in oxidative stress following TBI may disturb the balance of the reactive carbonyl species metabolism and lead to carbonylation of cytoskeletal proteins.

Since protein carbonylation is known to affect cytoskeleton stability, it can be suggested that the observed changes in cytoskeletal protein carbonylation in PC12 cells treated with H₂O₂ have physiological consequences. It has been reported that carbonylation of tubulin leads to the disassembly and instability of microtubules, while actin filaments are easily depolymerized upon carbonylation (19). Thus, in the present study, a series of assays were performed to examine the functional consequences of protein carbonylation. As shown in Fig. 2A and B, the results identified that the monomer/polymer ratios of β-actin and β-tubulin were significantly (P<0.05) increased with H₂O₂ treatment, which suggested dysfunction in the formation of polymers by the carbonylated β-actin and β-tubulin. These findings indicated that cytoskeletal proteins could not form stable polymer conditions in PC12 cells exposed to H₂O₂, providing evidence of the functional disturbance in cytoskeletal proteins under oxidative stress conditions.

The administration of AG at 0.5 h prior to H₂O₂ treatment resulted in reduction in the level of protein carbonylation when compared with that in the control group (Fig. 3A). In addition, the pretreatment with AG was found to also block the proportion of carbonylated β-actin and β-tubulin in PC12 cells exposed to H₂O₂. The quantification of the western blot analysis is shown in Fig. 3B, with a significant difference (P<0.05) observed between the H₂O₂-treated and H₂O₂+AG-treated groups.

It has been reported that reduced proteasomal activity contributes to the accumulation of carbonylated proteins during chronic experimental autoimmune encephalomyelitis in C57BL/6 mice (11). The proteasome serves a critical role in protein degradation and signal transduction following cellular stress or tissue injury (20). Overexpression of proteasome β5 assembled subunit increases the amount of proteasome and confers ameliorated response to oxidative stress and higher survival rates (21). In order to reveal the effect of proteasome β5 on the protein carbonylation, plasmids of proteasome β5 were transfected into PC12 cells. Upregulation of proteasome β5 in was first observed in the transfected PC12 cells (Fig. 4A and B). In addition, the level of protein carbonylation was increased compared with the control group. The western blot analysis results revealed that the overexpression of proteasome β5 in PC12 cells also blocked the proportion of carbonylated β-actin and β-tubulin following cell exposure to H₂O₂ (Fig. 4C and D).