Horseradish peroxidase labeled igg antibodies

After the whole brains were isolated, the hippocampus, hypothalamus, and pituitary gland were immediately dissected while the brains were placed on an ice surface. The tissues were stored at −80°C until further use. The SH-SY5Y cells were seeded into a six-well culture plate at a density of 2 × 10⁵ cells/well for 24 h; they were pretreated with SOCG (1, 10, or 100 μg/ml) for 1 h and then treated with CORT (100 μM) for 24 h. They were then rinsed with ice-cold PBS and lysed in the RIPA buffer. Equal amounts of each protein sample were resolved on 8–18% sodium dodecyl sulfate-polyacrylamide gels; the resultant bands were transferred onto nitrocellulose membranes (Hybond ECL; Amersham Pharmacia Biotech, Piscataway, NJ, USA). The membranes were blocked in 5% skim milk solution for 1 h. Next, they were incubated with antibodies against BDNF, CREB, GR (1 : 1000; Santa Cruz Biotechnology Inc., CA, USA), ERK (1 : 1000; Cell Signaling Technology, MA, USA), and nNOS (1 : 1000; Merck, Darmstadt, Germany) overnight at 4°C and then with horseradish peroxidase-labeled IgG antibodies (1 : 2000; Santa Cruz Biotechnology Inc., CA, USA) for 2 h at room temperature. For the detection of the protein bands, the ECL Western Blotting Detection System (Amersham Biosciences, PA, USA) was used. The band intensities were measured using the ImageJ software (version 1.49).

The SH-SY5Y cells were seeded into a six-well culture plate at a density of 2 × 10⁵ cells/well for 24 h and then treated with TGF-β (1–20 ng/mL) for 24 h. They were then rinsed with ice-cold PBS and lysed in the RIPA buffer. Equal amounts of each protein sample were resolved on 8–18% sodium dodecyl sulfate-polyacrylamide gels; the resultant bands were transferred onto nitrocellulose membranes (Hybond ECL; Amersham Pharmacia Biotech, Piscataway, NJ, USA). The membranes were blocked in 5% skim milk solution for 1 h. Next, they were incubated with antibodies against TH (1:1000; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and TGF-β receptor (1:1000; Abcam, Cambridge, UK) overnight at 4 °C, and then with horseradish peroxidase-labeled IgG antibodies (1:2000; Santa Cruz Biotechnology Inc.) for 2 h at room temperature. For the detection of the protein bands, the ECL Western Blotting Detection System (Amersham Biosciences, Little Chalfont, UK) was used.