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Fig. 5. The effect of specific β-isoform knockdown by RNAi on the amount of other isoforms. siRNA for β1 or β3 or both was introduced into HeLa cells. (A) After 24 hours, the cells were either directly harvested as the total cell lysate or harvested after the surface labeling with sulfo-NHS-SS-biotin as described in Fig. 2. The total cell lysate (whole cell) and the affinity-purified biotinylated proteins (surface) were analyzed by immunoblotting using 
1-, β1- or β3-specific antibodies. A typical result of the immunoblot is shown. The positions of the molecular size markers (in kDa) are indicated on the right. (B) The band intensity in A was quantified and represented as the ratio to that of the nontransfected cells. More than three independent experiments were performed to obtain error bars (s.d). (C) The amount of Na+/K+-ATPase subunits in the plasma membrane in β1/β3-double-knockdown cells. The siRNAs for both β1- and β3-subunits were introduced into the HeLa cells. After the indicated time, the cell surface proteins were labeled with biotin and purified. These samples were subjected to immunoblot analysis as in A and the amount of the 1-, β1- and β3-subunits were quantified and represented relative to the amount at the beginning of the chase. The loading volumes were adjusted based on the total protein amount in the sample. The results from three independent experiments are summarized (mean ± s.d.).
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