Rescue of A531V protein expression with a proteasomal degradation blocker.

<p>Characterization of CLC-1 protein turn-over in HEK293T cells. <i>(</i><b><i>A</i></b><i>)</i> Kinetics of protein degradation for WT CLC-1 and A531V in the presence of cycloheximide (100 µg/ml). <i>(</i><b><i>B</i></b><i>)</i> Quantification of CLC-1 protein expression levels in response to different cycloheximide treatment durations. Protein densities were standardized as the ratio of the myc-CLC-1 signal to the cognate β-actin signals, followed by normalization to those of the control at 0 hr. Data were averaged from 8 independent experiments. <i>(</i><b><i>C</i></b><i>)</i> The effect of treatment with 20 µM MG132. <i>(</i><b><i>D</i></b><i>)</i> Quantification of CLC-1 protein expression levels in response to different MG132 treatment durations. The scanned intensities of protein densities were normalized to those of WT with no drug treatment. <i>(</i><b><i>E</i></b><i>)</i> The relative expression ratio of A531V with respect to WT (as calculated from D) was plotted against the duration of the MG132 treatment. <i>(</i><b><i>F</i></b><i>)</i> Ubiquitination of CLC-1 proteins. Transfected cells were incubated at 37°C for 24 hrs in the presence of MG132. Cell lysates were immunoprecipitated (IP) with the anti-myc antibody, followed by immunoblotting (WB) with the anti-myc or anti-ubiquitin (Ub) antibody. Corresponding expression levels of CLC-1 constructs in the lysates are shown in the <i>Input</i> lane, which represents 5% of the total protein used for immunoprecipitation. Ub-CLC-1: ubiquitinated CLC-1.