14-3-3θ is a Binding Partner of Rat Eag1 Potassium Channels
Figure 9
Lack of effect of 14-3-3θ over-expression on the total and surface expression of rEag1 protein.
(A) Representative result of surface biotinylation experiments. Intact HEK293T cells were biontylinated on ice and thereafter solubilized. (Surface) Cell lysates were pulled down with streptavidin agarose beads, followed by immunoblotting with the anti-rEag1 antibody. (Input) Cell lysates were directly employed for immunoblotting analyses. Input represents 5% of the total protein used for streptavidin pull-down. Also shown at the bottom are the corresponding β-actin expression levels for each lane. The specificity of the biotinylation procedure was verified by the absence of β-actin bands in the surface fraction. (B) Quantification of total and surface expression of rEag1 in the absence or presence of 14-3-3θ over-expression. The total protein density (top panel) was determined as the ratio of input signal to the cognate β-actin signal. The surface expression efficiency (bottom panel) was expressed as the ratio of surface signal to the corresponding total protein density. The mean values were subsequently normalized with respect to that of vector control. Densitometric scans of immunoblots were obtained from three independent experiments.