Las 4000 chemiluminescence detection system

Immunoblot analyses were performed using polyclonal anti-PER1, monoclonal anti-hnRNP Q (Sigma-Aldrich), monoclonal anti-PTBP1 (Abcam), monoclonal anti-YBX1 (Abcam), polyclonal anti-hnRNP K (Abcam), monoclonal anti-hnRNP A1 (Abcam), monoclonal anti-FLAG (Sigma-Aldrich), polyclonal anti-14-3-3ζ (Santa Cruz Biotechnology) and monoclonal anti-GAPDH (Millipore) as primary antibodies. Horseradish peroxidase-conjugated species-specific secondary antibodies (goat, Santa Cruz Biotechnology; guinea pig, Santa Cruz Biotechnology; mouse, Thermo Fisher Scientific; rabbit, Jackson ImmunoResearch Laboratories) were visualized by using a SUPEX ECL solution kit (Neuronex) or a Pierce^TM ECL Western Blotting substrate (Thermo Fisher Scientific) under LAS-4000 chemiluminescence detection system (FUJIFILM) and a FUSION Solo S (Vilber). The acquired images were analyzed according to the manufacturer’s instructions.

Isolated hippocampal tissues were homogenized, and the concentration of the isolated proteins was then determined using a bicinchoninic acid (BCA) protein assay kit (Thermo, Rockford, IL, USA) (n = 3–5 per group). The homogenates (25 μg of protein) were subsequently separated by SDS-PAGE and transferred to PVDF membranes. The membranes were then blocked in 5% (w/v) skim milk to reduce nonspecific binding and probed with various primary antibodies, as listed in Table 1. The secondary antibodies corresponded to the type of primary antibody and included horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG (1:10,000, Amersham Pharmacia Biotech, Piscataway, NJ, USA). Protein bands were visualized using an enhanced chemiluminescence detection system (Amersham). Protein bands were quantified using an LAS-4000 chemiluminescence detection system (Fujifilm, Tokyo, Japan). The target protein density was normalized to β-actin levels, and protein level changes in the brain tissues of mice treated with footshock are presented relative to the control group. The changes in protein levels refer to control levels to compare experimental conditions.

Harvested cells were lysed in buffer containing 4% SDS and 2 M urea in phosphate-buffered saline (PBS). Western blotting and dot blotting were performed as we previously described^{48 (link)}. Antibodies were purchased as indicated: anti-EGFR (Abcam, Cambridge, MA), anti-GAPDH (ICN Biomedicals, Irvine, CA), anti-phospho-ERK (Santa Cruz Biotechnology, Dallas, TX), anti-14-3-3ξ (Santa Cruz Biotechnology, Dallas, TX), anti-EGF (Abcam, Cambridge, MA), anti-BAX (Cell Signaling Technology, Danvers, MA), anti-p53 (Santa Cruz Technology, Dallas, TX), anti-γH2AX (Cell Signaling Technology, Danvers, MA). HRP-conjugated species-specific secondary antibodies (KPL, Gaithersburg, MD) were visualized under LAS-4000 chemiluminescence detection system (FUJIFILM, Tokyo, Japan). Acquired images were analyzed using Image Gauge (FUJIFILM, Tokyo, Japan) according to the manufacturer’s instruction.

Cell extract and protein preparation, immunoprecipitation, and Western blotting were performed as previously described9 (link)14 (link). Protein bands were visualized using a SUPEX ECL solution kit (Neuronex, Daegu, South Korea) and a LAS-4000 chemiluminescence detection system (Fujifilm, Tokyo, Japan).

After starvation for 12 h in serum-free medium, cells were seeded into six-well plates and treated with test compounds. Total proteins were extracted using a RIPA lysis buffer (50 mM Tris–HCl (pH 8.0), 1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl and 1 mM phenylmethylsulfonyl fluoride) and subjected to western blot analysis with specific antibodies. The proteins were then visualized by enhanced chemiluminescence (Intron, Kyunggi, Korea) and detected using an LAS4000 chemiluminescence detection system (Fuji, Tokyo, Japan).

The protein was extracted from the hippocampus of the mice (n = 3–5 per group). The amount of protein was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce). The protein (50 μg) was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The blots were probed with various primary antibodies as listed in Table 1. The same blot was probed for the housekeeping protein β-actin, which served as a loading control. Secondary antibodies, including anti-rabbit IgG HRP-conjugated antibody (1:10,000; Amersham Pharmacia Biotech; Piscataway, NJ, USA) and anti-mouse IgG HRP-conjugated antibody (1:10,000; Amersham Pharmacia Biotech; NJ, USA), were used. The specific antibody-antigen complexes were detected by an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech; NJ, USA). The quantification was performed using an LAS-4000 chemiluminescence detection system (Fujifilm; Tokyo, Japan), and the target protein density was normalized to that of the internal control β-actin.