Fig. 3

Difopein increases both immature and mature Eag1 protein levels. A Difopein co-expression promotes immature Eag1 protein stability in HEK293T cells. (Left) Representative immunoblot showing the effect of R18 mutant or difopein co-expression on Eag1 protein turn-over time course in the presence of brefeldin A (BFA). Transfected HEK293T cells were pretreated with BFA (10 μM) for 12 h, followed by cycloheximide (CHX) treatment for the indicated duration. (Center) Linear plot of Eag1 protein degradation time course in the presence of BFA treatment. (Right) Semi-logarithmic plot of linear-regression analyses (solid lines) of the same data points shown to the left. Protein densities were standardized as the ratio of Eag1 signals to the cognate GAPDH signals, followed by normalization with respect to the corresponding no-CHX-treatment (0 h) control. Data points represent the average of four independent experiments. B Difopein co-expression augments cell-surface Eag1 protein level. (Left panels) Representative immunoblots. Cell lysates from biotinylated intact cells were either directly employed for immunoblotting analyses (Total) or subject to streptavidin pull-down prior to immunoblotting analyses (Surface). Actin was used as the loading control. (Right panels) Quantification of total and surface protein levels, as well as membrane trafficking efficiency (Surface/total). Total and surface protein densities were standardized as the ratio to the cognate total actin signal, followed by normalization with that of the R18 mutant control. Membrane trafficking efficiency was calculated as surface protein density divided by the corresponding standardized total protein density, followed by normalization with respect to the surface/total ratio of the R18 mutant control (*, P < 0.05; n = 3)