SDS 10% (v/v) glycerol) and equal amounts of cell protein (30 g/well) were fractionated by SDSCPAGE and electro-blotted onto nitrocellulose membranes Protein loading and electro-blotting efficiency were verified through Ponceau S staining and the membrane was blocked in Tween-Tris buffered saline (TTBS: 100 mM TrisCHCl pH 75 containing 09% NaCl and 01% Tween-20) containing 5% albumin Membranes were incubated blood vessel overnight at 4 C with each antibody separately in TTBS at different working dilutions as suggested by the manufacturers and then washed with TTBS Anti-rabbit IgG peroxidase-linked secondary antibody was incubated with the membranes for additional 1 h (1:5000 dilution range) washed again and the immunoreactivity was detected by enhanced chemiluminescence using ECL Plus kit Densitometric analysis of the films was performed with Image .

Quant software Blots were developed to be linear in the range used for densitometry All results were expressed as a relative ratio the antioxidant enzyme immunocontent and the  -actin internal control immunocontent 25 MTT assay Following retinol treatment Sertoli cells viability was assessed by the MTT assay This method is based on the ability of viable cells to reduce MTT (3-(45-dimethyl)- 25-diphenyl tetrazolium bromide) and form a blue formazan product MTT solution (sterile stock solution of 5 mg/ml) was added to the Bcr-Abl Inhibitors incubation medium in the wells at a final concentration of 02 mg/ml The cells were left for 45 min at 37 C in a humidified 5% CO2 atmosphere The medium was then removed and plates were shaken with DMSO for 30 min .

The optical density of each well was measured at 550 nm (test) and 690 nm (reference) H2O2 1 mM was used as positive control for cell death An in vitro control experiment was performed with varying concentrations of retinol (1C20 M) incubated for varying times with MTT (02 mg/ml) but no alterations on absorbance have been observed (not shown)As previously observed 24 h retinol incubation is able to enhance cellular reactive species production at 7 and 14 M (Fig 1A) As cellular viability is compromised by retinol 14 M we conducted further experiments using retinol at 7 M as this concentration was able to AZD2171 increase ROS production but at the end of the treatment cells were still viable according MTT results (Fig 1B) We have previously observed that pro-oxidant concentrations of retinol increase the immunocontent of RAGE in Sertoli cells after 24 h of incubation (Gelain et al 2008a) Here we tested the effect of inhibition of different protein kinases to determine the role of different signal pathways in this effect We used a range of specific pharmacological inhibitors that are widely used to block the activity of different protein kinases The concentration of the inhibitors was chosen based on what is recommended by the literature to effectively block each protein kinase activity with optimal specificity in non-cancer cultured cells (Dar and Shokat 2010; Gelain et al 2006 2007;