H 119

Inhibitors of ERK kinase (U0126), p38 (SB203580), NF-κB (BAY117082), and Akt (LY294002) were purchased from Calbiochem (Billerica, MA,USA) and were dissolved in dimethyl sulfoxide. Antibodies against phospho-ERK and phospho-p38 were purchased from Cell Signaling Technology (Danvers, MA, USA), and antibodies against phospho-Akt, NF-Kb p50 (H-119) and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Forklift-generated diesel exhaust particulates (SRM 2975, U.S. National Institute of Standards and Technology, MD, USA) were used in this study.

Frozen tissues were homogenized in a solution containing 0.5% hexadecyl-trimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 4,000 ×g at 4°C. Tissues protein concentration was measured by the Bradford method (1976); then, 15 μg protein sample was used for the gel electrophoresis in a 6% PAGE separation gel. The samples were electrotransferred onto a PVDF membrane. Blots were blocked with 5% nonfat dry milk for 1 h at room temperature and then incubated with primary specific antibodies overnight, followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The signal was normalized to the intensity of a housekeeping protein and expressed as densitometric unit (DU). Western Blots were performed to evaluate the expression of the UPS system (20S and 26S proteasome subunits), NF-κB (p50, p65, and p105 subunits). The following primary antibodies purchased by Santa Cruz (USA) were used: anti-proteasome subunit (Fl-76, anti 20S, and anti 26S), NF-κB p65 (C-20), NF-κB p50, and p105 (H-119). For all assays secondary antibodies HRP horseradish peroxidase were used: donkey polyclonal-rabbit IgG, goat anti-mouse, goat anti-rabbit, and were all purchased by Santa Cruz (USA).

The primary antibodies utilized were: rat anti-serotonin antibody (1:100 dilution, clone YC5/45, Merck), rabbit anti-ankyrin-G (anti-ankG; 1:300 dilution, H-215, Santa Cruz Biotechnology), mouse anti-microtubule-associated protein-2 (anti-MAP-2; 1:250 dilution, A-4, Santa Cruz Biotechnology), goat anti-choline acetyltransferase (anti-ChAT; 1:100 dilution. AB144P, Merck Life Science A/S), rabbit anti-5-HT_1A (1:100, H-119, Santa Cruz Biotechnology), rabbit anti-5-HT_1A (1:50, ASR-021, Alomone Labs), rabbit anti-5-HT_1A (1:100, ADI905-741-100, Enzo Life Sciences), rabbit 5-HT_1A (1:100, NB100-92418, Novus Biologicals), and mouse anti-HA epitope (1:500 dilution, clone 16B12, Biolegend). Rabbit 5HT_1A S1A-170 serum (1:1000–1:2500 dilutions) was a generous gift from Professor Efrain C. Azmitia, New York University, New York, NY, USA.