Gr m 20

Western blot analysis of protein targets was performed as previously described (Tai et al. 2002 (link)(Tai et al. , 2007 (link)(Tai et al. , 2009)) (link). Briefly, 50 mg of total protein per sample was resolved on 12% SDSpolyacrylamide gels and subsequently transferred to PVDF membranes (BioRad) in transfer buffer (48 mM Tris, 39 mM glycine, 0.037% SDS, 20% methanol) at 100V for 1 h. Transfer and equal loading of protein were verified by Ponceau S stain (Sigma). Membranes were incubated for 1h at 48C in blocking solution containing 5% skim milk in Tris-buffered saline with Tween-20 (TBST; 10 mM Tris-HCl, 150mM NaCl, 0.05% Tween-20), then rinsed in TBST (3!10 min) before overnight incubation with primary antibodies specific for rat PNMT (Immunostar, Hudson, WI, USA; 1:3000 dilution), Egr-1 (C-19, Santa Cruz Biotechnologies; 1:1000), Sp1 (PEP-2, Santa Cruz; 1:2500), GR (M-20, Santa Cruz; 1:2000), AP-2 (Millipore, Billerica, MA, USA, 1:2000) in 5% skim milk-TBST. Membranes were then rinsed in TBST (3!10 min) and incubated with HRP-conjugated secondary IgG (Sigma; 1:5000) in 5% skim milk-TBST for 1 h, followed by a final series of rinses in TBST (3!10 min). Proteins were then detected by enhanced chemiluminescence (Haan & Behrmann 2007) (link) and subsequent exposure to film (Pierce, Rockford, IL, USA).