Gel image analysis system

Total RNA was extracted from the CA1 region with use of the RNA Quick Extraction Kit (AXYGEN). Primers were purchased from BGI. RNA (1 μg of total) was used as a template for reverse transcription with use of the Toyobo Kit and amplified with TransGen Mix. PCR products were analysed using electrophoresis on a 1.5% agarose gel and were assessed using the Gel Image Analysis System (Bio‐Rad).

The lysed cell homogenates were centrifuged at 13,000 ×g for 10 min at 4°C to remove cell debris. Total proteins were collected and the protein concentrations were determined using the Bradford method. Proteins (20 μg) were separated on 10% SDS-PAGE and transferred to polyvinyl difluoride membranes (Millipore, Bedford, MA). Membranes were blocked with 5% milk in TBS-T (TBS containing Tween 20) for 30 min and incubated with primary antibodies overnight at 4°C. Primary antibodies were all obtained from Abcam (Cambridge, MA) and used at the following dilutions: hTERT (1 : 1000), phospho-JNK1 and phospho-JNK2 (pT183 and pY185, resp.; 1 : 1000), NK1/2 (1 : 500), phospho-c-Jun (pS63; 1 : 5000), c-Jun (1 : 1000), phospho-p38 (pT180; 1 : 1000), p38 (1 : 200), pro-caspase-9 (1 : 2000), cleaved caspase-9 (1 : 2000), pro-caspase-3 (1 : 1000), cleaved caspase-3 (1 : 500), GAPDH (1 : 10000), and β-actin (1 : 2500). After washing three times with TBS-T for 10 min, membranes were incubated with goat polyclonal anti-rabbit IgG-H&L-preadsorbed (HRP) secondary antibody at room temperature for 1 h. Protein bands were visualized by chemiluminescent detection and the densitometric values were determined using a gel image analysis system (Bio-Rad, Hercules, CA). Data were normalized to GAPDH or β-actin.

The treated cells were washed with phosphate-buffered saline (PBS) and disrupted in RIPA buffer mixed with phenylmethanesulfonyl fluoride and phosphatase inhibitor cocktails (Sigma) on ice. The protein concentrations were measured using a BCA Protein Assay kit (Pierce). The protein was then denatured with sample buffer (X5) for 10 minutes at 99˚C. The equivalent proteins were separated on a sodium dodecyl sulfate–polyacrylamide gel and then transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The PVDF membranes were blocked with 5% bovine serum albumin–Tris-buffered saline and Tween 20 (TBST) for 2 h at room temperature and then incubated with the indicated primary antibodies overnight at 4˚C. After being washed with TBST, the membranes were incubated with secondary antibody for 2 h at 37˚C. The blots were scanned by the Gel Image Analysis System (Bio-Rad).