Nedd4 does not affect MAK-V protein level.

<p>(<b>A</b>) HEK293 cells were transfected with MAK-V-FLAG expression plasmid (<i>MAK-V-FLAG +</i>) together with plasmid for expression of <i>myc</i>-tagged wild-type Nedd4 (<i>wt</i>) or its catalytically inactive Nedd4(CS) mutant (<i>CS</i>), or mock-untransfected (-). Cells were treated by MG132 prior to lysis (<i>MG132+</i>) or left untreated (<i>MG132 -</i>). Results of Western blot analysis of total cell lysates with anti-FLAG antibodies are shown (<i>anti-FLAG</i>). Nedd4 levels were monitored with anti-Nedd4 antibodies (<i>anti-Nedd4</i>), and exogenously expressed myc-tagged Nedd4 proteins were detected with anti-<i>myc</i> antibodies (<i>anti-myc</i>). To monitor total protein loading, membrane was re-probed with anti-α-tubulin antibodies. (<b>B</b>) HEK293 cells were co-transfected with MAK-V-FLAG expression plasmid and plasmid for expression of <i>myc</i>-tagged wild-type Nedd4 (<i>Nedd4 wt</i>) or its catalytically inactive Nedd4(CS) mutant (<i>Nedd4(CS</i>)). Cells were treated with cycloheximide for indicated time prior to lysis. Representative results of Western blot analysis of total cell lysates with anti-FLAG antibodies to detect MAK-V-FLAG protein are shown (<i>anti-FLAG</i>). To monitor total protein loading, membrane was re-probed with anti-α-tubulin antibodies. (<b>C</b>) Quantification of MAK-V-FLAG protein level in HEK293 cells when co-expressed with wild-type Nedd4 (<i>Nedd4 wt</i>) or with its Nedd4(CS) (<i>Nedd4 CS</i>) mutant after cycloheximide treatment. Relative MAK-V-FLAG protein levels for each time point of cycloheximide treatment indicated in hours (<i>hrs</i>) were determined by quantification of Western blot images obtained as described in (<b>B</b>). Data are presented as means ± standard deviation of three independent experiments performed as described in (<b>B</b>).