Figure 4. USP7–DNMT1 interaction is required for USP7-mediated stabilization of DNMT1.

(a) DNMT1 protein levels in HEK293T cells stably expressing USP7 shRNAs. The protein levels were determined by immunoblotting, and the messenger RNA levels were determined by quantitative real-time PCR. The error bars represent ±s.d. from triplicate experiments. Uncropped blots are shown in Supplementary Fig. 7. (b) Effect of USP7–DNMT1 interaction on ubiquitination of DNMT1. HEK293T cells were cotransfected with either wild-type or mutant RFP–USP7 and HA–ubiquitin, followed by immunoprecipitation of DNMT1. Polyubiquitination was detected by immunoblotting with HA antibody. As input, the whole cell lysates were analysed by immunoblotting using the indicated antibodies. Uncropped blots are shown in Supplementary Fig. 7. (c) Effect of USP7–DNMT1 interaction on DNMT1 protein stability. HEK293T cells were infected with GFP-tagged wild-type and mutant USP7 lentivirus. The protein levels of DNMT1, USP7 and β-actin were determined (top), and the relative protein level of DNMT1 was quantified (bottom). The error bars represent ±s.d. from triplicate experiments. Uncropped blots are shown in Supplementary Fig. 7. (d) Protein stabilities of DNMT1 and DNMT1^4KQ. HEK293T cells stably expressing DNMT1 and DNMT1^4KQ were treated with CHX (100 μg ml⁻¹) and harvested at the indicated time points. The expression levels of DNMT1 and β-actin were determined (top), and the relative DNMT1 protein level was quantified (bottom). Uncropped blots are shown in Supplementary Fig. 7.