Fig. 1

Difopein increases Eag1 protein expression. A (Left) Representative immunoblot showing the effect of difopein co-expression on Eag1 over-expressed in HEK293T cells. Cells were transfected with cDNAs for Eag1, as well as YFP vector, YFP-difopein, or YFP-R18 mutant (YFP-R18 mut). Two days post-transfection, cells were subject to immunoblotting analyses with the anti-Eag1 (α-Eag1), anti-GFP (α-GFP), and anti-β-actin (α-actin) antibodies. (Right) Quantitative analyses of relative Eag1 protein levels for the three co-transfection conditions. Protein densities were standardized as the ratio to the cognate β-actin signals, followed by normalization with respect to the YFP vector control (*, P < 0.05; n = 5). B Lack of effect of difopein co-expression on Eag1 mRNA level in HEK293T cells. Semi-quantitative RT-PCR analyses of relative Eag1 mRNA levels were employed for the three co-expression conditions. mRNA levels of Eag1 were standardized as the ratio of Eag1 signals to the cognate GAPDH mRNA levels, followed by normalization with respect to the YFP vector control (n = 3). C–D Effects of shRNA knockdown of various endogenous 14-3-3 proteins on Eag1 protein (C) or mRNA (D) levels in HEK293T cells. HEK293T cells over-expressing Eag1 were subject to infection with a control shRNA for GFP (sh-GFP), or shRNA specific for 14-3-3β, η, or θ isoforms (sh-14-3-3β#1, sh-14-3-3β#2, sh-14-3-3η, sh-14-3-3θ#1, sh-14-3-3θ#2). Quantitative analyses of relative Eag1 protein levels are based on normalization with respect to the sh-GFP control (*, P < 0.05; n = 4). Lack of effect of 14-3-3 knockdown on Eag1 mRNA levels is supported by quantitative analyses of relative Eag1 mRNA levels normalized with respect to the sh-GFP control (n = 5)