Rescue of A531V protein expression with a proteasomal degradation blocker.

Characterization of CLC-1 protein turn-over in HEK293T cells. (A) Kinetics of protein degradation for WT CLC-1 and A531V in the presence of cycloheximide (100 µg/ml). (B) 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. (C) The effect of treatment with 20 µM MG132. (D) 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. (E) The relative expression ratio of A531V with respect to WT (as calculated from D) was plotted against the duration of the MG132 treatment. (F) 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 Input lane, which represents 5% of the total protein used for immunoprecipitation. Ub-CLC-1: ubiquitinated CLC-1.