Fig. 5

Difopein up-regulates Eag1 protein expression in neurons. A, B Over-expression of difopein promotes endogenous Eag1 expression in cultured cortical neurons. Neurons (DIV10) were transfected with YFP, YFP-difopein, or YFP-R18 mutant, and then incubated for two days, followed by immunoblotting (A) or immunofluorescent (B) analyses. Quantitative analyses of immunoblots are summarized by the bar graphs. Protein densities were standardized as the ratio to the cognate GAPDH signals, followed by normalization with respect to the YFP vector control (*, P < 0.05; n = 5). Endogenous Eag1 was detected with the anti-Eag1 antibody (red), and YFP-tagged proteins were directly visualized (green). Arrowheads denote punctate Eag1 staining patterns. Scale bar, 10 μm. See Additional file 1: Fig. S4 for further quantitative analyses of immunofluorescent images. C shRNA knockdown of 14-3-3β and 14-3-3θ, but not 14-3-3η, increases endogenous Eag1 protein level in cultured cortical neurons. Neurons (DIV10) were infected with various viral stocks and selected with puromycin; two days post-infection, immunoblotting analyses were performed using the indicated antibodies