Figure 5

Gα o promotes Necdin-induced neurite outgrowth. (A) Neuro2a cells were transfected with plasmids encoding various types of Gα (0.5 μg) and FLAG-Necdin (1 μg). To identify transfected cells, we co-transfected with the pEGFP (100 ng). After 24 h of transfection, cells were serum-starved and observed 30 h later. The proportions of neurite-bearing cells were determined, as described in Materials and Methods section. Data are presented as the average ± SE of at least three independent experiments. *, p < 0.001 (B) Left panel, Lysates of Neuro2a cells expressing plasmids encoding FLAG-Necdin (10 μg), E2F1 (0.3 μg), and GαoQ205L (1 μg or 3 μg) were subjected to an immunoprecipitation assay using anti-FLAG antibodies, and the beads analyzed with antibodies against E2F1, FLAG, and Gαo. Right panel, Neuro2a cells were transfected with plasmids encoding FLAG-GαWT (10 μg), HA-Necdin (20 μg), and E2F1 (1 μg). Cell lysates were immunoprecipitated with anti-Necdin antibodies. Immunocomplexes were subjected to immunoblot analysis using antibodies against E2F1, Necdin, and FLAG. Input was loaded with 10% of Neuro2a cell extracts used for the immunoprecipitation assay. The numbers beside blot indicate size marker (kDa). (C) Neuro2a cells were transfected with the indicated combinations of plasmids encoding GαoQ205L (0.2 μg), FLAG-Gαo/i (0.2 μg), FLAG-Necdin (0.25 μg), E2F1 (0.03 μg), E2F4B-Luc reporter gene (0.1 μg), and β-galactosidase (0.3 μg). The total amount of plasmid DNA used for transfection was maintained by adding pcDNA3. After 48 h, cells were subjected to luciferase and β-galactosidase assays. Luciferase activity was normalized to that of β-galactosidase. Data are presented as the average ± SE of at least three independent experiments. *, p < 0.05; **, p < 0.001.