Fig. 2

Difopein promotes Eag1 protein stability. A Difopein co-expression enhances Eag1 protein half-life values in HEK293T cells. (Upper left) Representative immunoblots showing the protein turn-over time course of Eag1 co-expressed with R18 mutant or difopein in HEK293T cells. 48 h post-transfection, cells were subject to 100 μg/ml cycloheximide (CHX) treatment for the indicated durations. (Lower left) Linear plot of relative Eag1 protein levels in response to different CHX treatment durations. Protein densities were standardized as the ratio of Eag1 signals to the cognate GAPDH signals, followed by normalization with respect to the no-CHX-treatment (0 h) control. (Lower right) Semi-logarithmic plot of linear-regression analyses (solid lines) of the same data points shown to the left. (Upper right) Statistical comparisons of Eag1 protein half-life values for the two co-expression conditions (*, P < 0.05; n = 3). B, C Difopein co-expression diminishes Eag1 protein ubiquitination. Transfected cells were treated with 10 μM MG132 (B) or 100 μM chloroquine (CQ) (C) for eight hours, followed by immunoprecipitation (IP) with the anti-Eag1 antibody. (Top panels) Representative immunoblot showing Eag1 ubiquitination in the absence or presence of difopein co-expression. Ubiquitinated Eag1 is visualized as high-molecular-weight protein smears detected with the FK2 anti-ubiquitin antibody. (Bottom panels) Quantification of relative ubiquitinated Eag1 levels normalized with respect to the R18 mutant control (*, P < 0.05; n = 3). D, E Difopein co-expression abolishes MG132- (D) and CQ- (E) induced increase in Eag1 protein level. (Left panels) Representative immunoblots. (Right panels) Quantitative analyses of the effect of MG132 (D) and CQ (E) treatments on Eag1 protein levels for the two co-expression conditions. Protein densities were standardized as the ratio to the cognate GAPDH signals, followed by normalization with respect to the corresponding vehicle-treated control (*, P < 0.05; n = 6)