Isolated colonies were allowed to grow. C/EBP β expression was tested by western blotting in at least 10 different clones per plasmid transfection. Selected clones were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mm glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin, and 500 μg/mL G418 antibiotic (all chemicals were from Sigma-Aldrich). For western blot analysis of C/EBP β expression in CGNs, both samples from nucleo-cytosol separation

and total protein were used. To separately extract nucleic and cytosolic proteins from CGNs (Caruccio & Banerjee, 1999), CGN cell cultures from 12 rat pups were scraped, resuspended in 150 μL of extraction buffer with low salt [20 mm HEPES, pH 7.9, 10 mm NaCl, 3 mm MgCl2, 0.1% this website NP-40, 10% glycerol, 0.2 mm EDTA, 1 mm dithiothreitol (DTT), protease and phosphatase inhibitor cocktails] and left Birinapant on ice for 10–15 min with occasional tapping. Nuclei were pelleted by centrifugation at 700 g for 5 min at 4 °C. The cytoplasmic supernatant fraction

was transferred into another Eppendorf tube. Nuclei were washed with 200 μL of washing buffer to remove NP-40 (20 mm HEPES, pH 7.9, 0.2 mm EDTA, 20% glycerol, 1 mm DTT, protease and phosphatase inhibitor cocktails), and centrifuged at 700 g for 5 min at 4 °C. Nuclei were then resuspended in 60 μL of extraction buffer with salt (20 mm HEPES, pH 7.9, 400 mm NaCl, 0.2 mm EDTA, 20% glycerol, 1 mm DTT, protease and phosphatase inhibitor cocktails), and left on ice for 45 min with periodic mixing by tapping in order to extract nuclear proteins. Following centrifugation at 14 500 g for 15 min at 4 °C, supernatants were removed, aliquoted, quickly frozen on dry ice, and stored at −80 °C. The cytoplasmic fraction was further clarified by adding a one-third

volume of cytoplasmic extraction clarification buffer (20 mm HEPES, pH 7.9, 400 mm NaCl, 0.2 mm EDTA, 40% glycerol, 1 mm DTT, protease and phosphatase inhibitor cocktails) for 30 min at 4 °C in order to equilibrate the cytoplasmic proteins with NaCl, and this was followed by centrifugation at 14 500 g for 15 min. In parallel, total samples were obtained by directly harvesting cell cultures in Laemli Sample Buffer. 4��8C Transfected CGNs and DAOY stable clones were lysed in lysis buffer (50 mm Tris, pH 7.4, 1 mm EDTA, 1% sodium dodecyl sulfate, protease inhibitors), and then sonicated with a tip sonicator for 5 s at 10% power output. Total protein sample content was determined with the Lowry quantification method (Lowry et al., 1951), and 30 μg of each sample was loaded per lane for western blot analysis. Samples obtained as previously described were briefly sonicated and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis before electroblotting. Membranes were incubated with antibodies against C/EBP β [rabbit (C-19) C-terminal rat epitope; Santa Cruz Biotechnology, Santa Cruz, CA, USA; cat. no.