Figure 1
CUL4A and 4B control the protein level of CLC-1.
Biochemical demonstration of the regulation of CLC-1 by cullin in HEK293T cells.(A)(Left) Representative immunoblots showing the effect of 24-hr treatment of 10 μM MLN4924 (in 0.1% DMSO) on protein expression of Myc-CLC-1. The molecular weight markers (in kilodaltons) and immunoblotting antibodies (α-Myc and α-actin) are labeled to the left and right, respectively. Expressions of actin are displayed as the loading control. (Right) Quantification of relative CLC-1 protein expression level. Protein density was standardized as the ratio of the CLC-1 signal to the cognate actin signal. Values from the MLN4924-treated group (hatched bars) were then normalized to those for the corresponding control (clear bars). Asterisks denote significant difference from the control (*, t-test: p < 0.05; n = 5-6).(B) The effect of Flag-DN-CUL4A/B co-expression on CLC-1. Co-expression with the Flag vector was used as the control experiment (Ctrl). Tubulin was used as the loading control. (C) Quantification of relative CLC-1 protein level in the presence of various DN-CUL constructs. See Supplementary Figure S1 for more immunoblots. Asterisks denote significant difference from the Flag vector control (Ctrl) (*, t-test: p < 0.05; n =4-16). (D)(Top) The kinetics of CLC-1 protein turn-over in the presence of different treatment durations of 100 μg/ml cycloheximide (CHX). Co-expression with the pcDNA3 vector was used as the control experiment. (Bottom) Protein densities were standardized as the ratio of CLC-1 signals to the cognate actin signals, followed by normalization to those of the vector control at 0 hr. Based on linear-regression analyses of the semi-logarithmic plot of the protein degradation time course, the protein half-life values for CLC-1 WT (n = 7-20) are about 6.1 (Control), 9.5 (DN-CUL4A) and 10.8 (DN-CUL4B) hrs; those for the A531V mutant (n = 8-15) are about 3.3 (Control), 5.6 (DN-CUL4A) and 6.5 (DN-CUL4B) hrs.(E) The effect of shRNA knock-down of endogenous CUL4A/B. The numbers denote the relative CLC-1/CUL4A/CUL4B expression level with respect to the control shRNA. The gels were run under the same experimental conditions. Uncropped images of immunoblots are shown in Supplementary Figure S2.
