Supplementary Figure 6: ZEB1 specifically regulates the protein stability of the USP7 target CHK1.

(a) qPCR of CHK1 in SUM159-P2 cells transduced with ZEB1 shRNA. n = 3 samples per group. (b) Quantification of CHK1 protein levels (normalized to GAPDH) in Fig. 5f. (c) Quantification of CHK1 protein levels (normalized to GAPDH) in Fig. 5g. (d) SUM159-P2 cells were transfected with the scramble control or USP7 siRNA (si-USP7) and then treated with 50 μg ml⁻¹ cycloheximide (CHX). Cells were harvested at different time points as indicated and then immunoblotted with antibodies to ZEB1, USP7 and GAPDH. (e) Quantification of CHK1 protein levels (normalized to GAPDH) in Fig. 5h. (f) SUM159-P2 cells were treated with 50 μg ml⁻¹ cycloheximide (CHX), harvested at different time points as indicated and then immunoblotted with antibodies to Claspin, ZEB1 and GAPDH. (g) 293T cells were transfected with SFB–USP7 alone or in combination with ZEB1, followed by pull-down with streptavidin-sepharose beads (s-s beads) and immunoblotting with the antibody to Claspin. Data in a are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Students t-test. The experiments were repeated 3 times. The source data can be found in Supplementary Table 3. Uncropped images of blots are shown in Supplementary Fig. 7.