, 2005); GAD65 forward primer 5′-GAA CAG CGA GAG CCT GGT-3′, reverse primer 5′-CTC TTG AAG AAG CTC ATT

GG-3′ (362 bp expected band size); TPH2 forward primer 5′–TAA ATA CTG GGC CAG GAG AGG-3′, reverse primer Trametinib purchase 5′-GAA GTG TCT TTG CCG CTT CTC-3′ (132 bp expected band size); and β-actin forward primer 5′-ATC CTG AAA GAC CTC TAT GC-3′, reverse primer 5′-AAC GCA GCT CAG TAA CAG TC-3′ (287 bp expected band size). The PCR conditions were conducted at 94 °C for 5 min, 30 cycles of 94 °C for 30 s, 60–65 °C for 1 min, 72 °C for 1 min followed by 10 min at 72 °C. The PCR products were then separated in 1.5% agarose gel, stained with ethidium bromide, and then visualised under UV irradiation. Real-time RT-PCR amplification on a separate group of sham- (n=4) and NI-lesioned (n=4) was carried out using the ViiA 7 Real-time RT-PCR system (Applied Biosystems, USA) with SYBR green PCR master mix (Applied Biosystems, USA) with the following gene-specific

primers: CRF1 forward primer 5′-ACG ACA AAC AAT GGC TAC CG-3′, reverse primer 5′-GCA GTG ACC CAGGTA GTT GA-3′; relaxin-3 forward primer 5′–CCC TAT GGG GTG AAG CTC TG-3′, reverse primer 5′-CCA GGT GGT CTG TAT TGG CT-3′; GAD65 forward primer 5′-GGG GTG GAG GGT TAC TGA TG-3′, reverse primer 5′-ACC CAT CAT CTT GTG GGG AT-3′; TPH2 forward primer 5′-GCC TTT GCA AGC AAG AAG GT-3′, reverse primer 5′-CGC CTT GTC AGA AAG AGC AT-3′; and β-actin forward primer 5′-ATC CTG AAA GAC CTC TAT GC-3′, reverse primer 5′-AAC GCA GCT CAG TAA CAG TC-3′. β-actin was used as an internal control. The PCR conditions were an initial incubation

GSK-3 signaling pathway of 50 °C for 2 min and 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 59 °C for 1 min. Fresh NI/MS tissue was dissected and lysed with a radioimmunoprecipitation assay (RIPA) buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris, pH 8.0). Proteins were first quantified with a Pierce bicinchoninic acid assay (BCA) kit (Thermo Scientific, USA), separated on a 12% sodium dodecyl sulphate (SDS)-PAGE and then transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 1% skim milk and incubated with a goat anti-CRF RI/II (1:1000, Santa Cruz, USA), rabbit anti-GAD65 (AB5082, 1:1000, Millipore, USA), rabbit anti-TPH2 Ureohydrolase (1:1000, Chemicon, USA) and rabbit anti-actin (A2066, 1:10 000, Sigma-Aldrich, USA) or 5% Bovine Serum Albumin (BSA) solution and incubated with mouse anti-RLX3 (1:2500) overnight at 4 °C. The blot was then washed and incubated with a HRP-conjugated anti-goat (1:5000), anti-rabbit IgG (1:5000) or goat anti-mouse Alexa Fluor 488 (1:5000) for 1 h at room temperature with agitation. The proteins were then detected with a chemiluminescence kit.