Figure 2.

USP5 interacts with CCND1 and downregulates its polyubiquitination level. (A,B) Myc-USP5 or EV plasmid (1 µg) was cotransfected with Flag-CCND1 or empty vector (EV) plasmid (1 µg) into HEK293T cells. After 24 hours of transfection, cells were collected for lysate preparation. Equal amounts of proteins (200 µg) were first subjected to immunoprecipitation (IP) with 2 µg of anti-Myc (A) or anti-Flag (B) antibody (Ab), and subsequent immunoblotting (IB) with the indicated Abs. (C) Direct protein-protein interaction between Myc-USP5 and endogenous CCND1 was assessed in the non-small cell lung cancer (NSCLC) cell lines A549 and H1299. Myc-USP5 or EV plasmid (1 µg) was transfected into A549 and H1299 cells for 48 hours. Cells were collected for lysate preparation. The cell lysates were then subjected to IP with anti-Myc or normal rabbit IgG (negative control) Ab and subsequent IB with the indicated Abs. (D) Flag-CCND1 plasmid (1 µg) was cotransfected with Myc-USP5 and HA-Ub plasmids (1 µg/each), with EV and HA-Ub plasmids (1 µg/each), or with EV plasmids (1 µg/each) into HEK293T cells for 24 hours. Cell lysates were then prepared for IP with anti-Flag Ab and subsequent IB analysis of HA (anti-HA, 1:5,000) and Flag with indicated Abs. (E) NSCLC cell lines A549 and H1299 were transfected with Myc-USP5 or EV plasmid (1 µg) for 48 hours. Cell lysates were prepared for IP with anti-CCND1 Ab followed by IB analysis of Ub (anti-Ub, 1:1,000) and CCND1 with indicated Abs. (F) SiUSP5 or siNC (150 nM) was transfected into H1299 cells. After 48 hours, cell samples were prepared for IP/IB as indicated above. IB analysis of input served as a positive control for all IP/IB assays.