P jnk

The expression levels of Ki-67, cleaved caspase-3, Beclin1, LC3B-II, CHOP, GRP78, p-JNK, and p-c-jun in tumor tissues were measured by IHC analysis according to the protocols previously described [25 (link)]. Briefly, 4-mm consecutive sections were deparaffinized in xylene, rehydrated in a graded ethanol series, and submerged in EDTA antigenic retrieval buffer for 15 min in a microwave oven. The sections were treated with 3% hydrogen peroxide in absolute methanol for 20 min to block endogenous peroxidase activity. Then, 5% BSA was applied for 15 min to prevent non-specific binding. The sections were incubated overnight at 4 °C with primary antibodies. Ki-67 (1:150), cleaved caspase-3 (5 μg/ml), Beclin1 (1:200), LC3B-II (1 μg/ml), CHOP (1:100), GRP78 (1 μg/ml), p-JNK (1:100) and p-c-jun (1:100) were purchased from Abcam (Cambridge UK). After incubation with the secondary antibody, the visualization signal was developed with 3,30-diaminobenzidine tetrachloride.

Protein extracts were boiled for 5 min in SDS sample buffer, separated using SDS-PAGE, and transferred onto polyvinylidene difluoride membranes (Immobilon™-P, MerckMillipore, Billerica, MA, USA). The membranes were probed with the following primary antibodies: rabbit anti-MMP-2 (1:1000; Novus Biologicals, Littleton, CO, USA), mouse anti-MMP-9 (1:300; Abcam, Tokyo, Japan), Akt (1:1000; Santa Cruz, California, USA), pAkt (1:1000; Santa Cruz), p65 NF-κB (1:1000; Santa Cruz), IкB-α (1:1000; Santa Cruz), ERK1/2 (1:1000; Abcam, Cambridge, UK), pERK1/2 (1:1000; Abcam), p38 (1:1000; Abcam), p-p38 (1:1000; Abcam), JNK (1:1000; Abcam), pJNK (1:1000; Abcam), c-Fos (1:500; Santa Cruz), c-Jun (1:500; Santa Cruz), iNOS (1:1000; Santa Cruz), and mouse anti-β-actin (1:10,000; Merck). All immunoreactive proteins were detected using ECL™ Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK).

After injecting an overdose of urethane, rat L4–L5 spinal segments were removed immediately, cut into ipsilateral and contralateral quadrants, frozen in liquid nitrogen and stored at −80 °C for further analysis. The samples were homogenized using an ultrasonic cell processor in SDS sample buffer containing proteinase inhibitors and PMSF. Equal amounts of the protein sample were separated by performing 10% tris–tricine SDS–PAGE and were transferred onto PVDF membranes (Millipore). The membranes were blocked with 5% nonfat milk for 2 h at RT. The membranes were incubated overnight at 4 °C with rabbit anti-IFN-γ antibody (1:500 dilution; Abcam), JNK (1:1000 dilution; Abcam), pJNK (1:1000 dilution; Abcam) or goat anti-GFAP antibody (1:10000 dilution; Sigma) and then with HRP-conjugated secondary antibodies (1:1000 dilution; Pierce) for 2 h at RT. Bands were developed using enhanced chemiluminescence (Pierce) and were visualized using ChemiDoc XRS system (Bio-Rad). All western blotting were performed at least 3 times, and similar results were obtained from all the analyses. The density of band area was quantified using Image Lab 3.0. A same-sized square was drawn around each band to measure its density, and the background of that band was subtracted. GAPDH expression was used as a loading control for protein expression.