Figure 2. CSN6 is critical in affecting p57 ubiquitination. (A) CSN6 increases p57 ubiquitination in a dose-dependent manner. 293T cells were transfected with the indicated plasmids and increasing CSN6 expressing plasmids. The proteasome inhibitor MG132 was added 6 h prior to cell harvesting. The amount of ubiquitinated p57 was analyzed by immunoprecipitation (IP) with anti-Flag followed by immunoblotting (IB) with anti-ubi. TCE, total cell extracts. (B) CSN6 haplo-insufficiency leads to p57 stabilization. Indicated WT and csn6+/− MEF cells were immunoblotted with anti-p57 or tubulin antibodies. (C) CSN6 haplo-insufficiency leads to decreased p57 ubiquitination. Indicated MEFs were treated with proteasome inhibitor MG132 for 6 h prior to cell harvesting. The amount of ubiquitinated p57 was analyzed by immunoprecipitation (IP) with anti-p57 followed by immunoblotting (IB) with anti-ubi. TCE, total cell extracts.