Figure 3.

Cyclin G2 forms complexes with ubiquitin, Skp1 and Skp2 and is regulated by Skp2. (A) OV2008 cells were transfected with p3xFLAG-CCNG2 plasmid and recovered in the presence of 50 μM MG-132 or lactacystin for 2 h. The Flag-tagged CCNG2 was immunoprecipitated, and Western blots were performed using antibodies against ubiquitin, Flag, and Skp1. (B) Detection of cyclin G2/ubiquitin/Skp2 complex in pulldown samples. Cells in 10-cm dish were transfected with 5 μg of CCNG2-V5, HA-ubiquitin (Ub), and Flag-Skp2 plasmids, either alone or in combination. Ni-NTA beads were used to pulldown CCNG2-V5. CCNG2, ubiquitin, and Skp2 were detected using antibodies against V5, HA, and Flag, respectively. (C) Effect of Skp2 and MG-132 on cyclin G2 levels. Cells in 10-cm dish were cotransfected with 5 μg of CCNG2-V5, HA-Ub, and Flag-Skp2 plasmids in the presence or absence of MG-132. Western blots were performed using anti-V5, anti-HA, and anti-Skp2 antibodies. (D) Overexpression of Skp2 decreased cyclin G2. Cells in six-well plate were transfected with 3 μg of CCNG2-V5 and Flag-Skp2 plasmid alone or in combination. Western blots were performed using antibodies against V5 and Flag for CCNG2 and Skp2, respectively. (E) Knockdown of Skp2 increased cyclin G2 level. Cells were cotransfected with CCNG2-V5 and Skp2-siRNA or control-siRNA (C-siRNA), and Western blots were performed using anti-V5 and anti-Skp2 antibodies. Skp2-siRNA decreased endogenous Skp2 levels and enhanced CCNG2-V5 levels.