Forty microliters of luciferase assay buffer [75 mm Tris-HCl, pH 7.8, 10 mm MgCl2, 2 mm ATP, 1 mm d-luciferin (Sigma-Aldrich)] was added to 20 μL of CGN lysate, vortexed, and read with a luminometer (TD-20/20; Turner Designs). Luminescence values were normalized against total protein content for each sample determined with the Lowry method. For immunocytochemistry, CGN cultures from 16 rat pups were fixed for 20 min in 4% paraformaldehyde at room temperature. Non-specific sites were blocked with 0.1% normal goat GSK-3 inhibitor serum and 3% BSA (both from Sigma-Aldrich) in PBS/0.1% Triton X-100 for 1 h at room temperature. After several washes, cells were incubated overnight at 4 °C with antibody against C/EBP β (Santa Cruz

Biotechnology), p-(Ser105)-C/EBP β (New England Biolabs), or SUMO-2/3 (Santa Cruz Biotechnology), and further incubated with the secondary antibodies anti-rabbit fluorescein isothiocyanate, anti-rabbit tetramethylrhodamine isothiocyanate or anti-mouse fluorescein isothiocyanate (Sigma-Aldrich) for 1 h 30 min at room temperature. After this, nuclei were stained with Hoechst 33258 (0.1 mg/mL; Sigma-Aldrich) for 5 min GSK2118436 clinical trial at room temperature.

All antibodies were diluted in PBS/0.1% Triton X-100/1% BSA. To quantify neuronal cell death, normal and condensed nuclei were counted after Hoechst staining in either total CGN cultures or CGNs transfected with one of the plasmids, by considering GFP-positive neurons as co-transfected. CGNs were fixed for 20 min with 4% paraformaldehyde in 0.1 m phosphate buffer (77 mm Na2HPO4, 23 mm NaH2PO4), washed in PBS, and incubated for 5 min at room temperature with 0.1 g/mL Hoechst Farnesyltransferase 33258 (all from Sigma-Aldrich). Stained cultures were photographed with a fluorescence microscope (Eclipse TE 2000-S microscope; Nikon, Tokyo, Japan) equipped with an AxioCam MRm (Zeiss, Oberkochen, Germany) digital camera, by use of a × 20 objective, and counting was performed in five randomly selected fields of each dish in two dishes per experiment

(Monti et al., 2001). The viability of DAOY cells was evaluated by the thiazolyl blue [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)] assay (Hansen et al., 1989). This method is based on conversion of the tetrazolium salt to a colored compound, a reaction that occurs only in viable cells, because the chemical reaction is carried out by mitochondrial dehydrogenases. DAOY stable clones were plated in 24-well plates at a density of 25 000 cells per well. Twenty-four hours later, the cell medium was replaced with fresh serum-free DMEM (supplemented with 2 mm glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin, and 500 μg/mL G418) containing 5 μm lactacystin. All chemicals were from Sigma Aldrich. After 24 h of incubation, MTT (Sigma-Aldrich) was added to the culture medium to a final concentration of 0.1 mg/mL. Following 1 h of incubation at 37 °C in the dark, the crystals formed were dissolved in 0.1 m Tris-HCl buffer (pH 7.