FIG 3.

SCML2 is required for the interaction of BMI1 with USP7. (A) Western blot analysis of the expression of SCML2, BMI1, USP7, p53, and RING1B after knockdown with a control siRNA (C), two different siRNAs targeting both SCML2A and SCML2B (siRNAs 1 and 2), and two different siRNAs targeting SCML2A (siRNAs 3 and 4) in HCT116 cells. Ponceau staining is shown as a loading control. (B) Western analysis of pulldown after immunoprecipitation of BMI1 (left) and RING1B (right) performed with nuclear extracts from HCT116 wild-type cells (WT) or USP7-KO cells (KO). (C) Same as for panel B, using nuclear extracts from HCT116 cells transfected with a control siRNA or siRNA 1 targeting both SCML2A and SCML2B (1). (D) 293T cells were transfected with a plasmid encoding HA-ubiquitin together with siRNA 1 for SCML2. After 2 days, the ubiquitinated material was pulled down from nuclear extracts with an anti-HA antibody. The pulled-down material was analyzed by Western blotting with antibodies against BMI1, HA, SCML2, or CDK2 as a loading control. Five percent of the input is shown, the arrows indicate ubiquitinated bands of BMI1, and the bar denotes polyubiquitinated BMI1. (E) Schematic representation of canonical PRC1 showing the interaction domains and the bridging model of SCML2 and USP7. (F) Model for the interaction of USP7 with RING1B in a subcomplex with RYBP.