Fig. 5

USP13 stabilizes MYC protein via its deubiquitinating enzyme activity. A Immunoblot analyses of HEK293T cells with or without USP13 overexpression were performed with the indicated antibodies B Western blot showing USP13 and FLAG (MYC) and β-actin in control and USP13 knockdown cells following transfection with an empty vector or FLAG-MYC. C Co-immunoprecipitation (Co-IP) of exogenous FLAG-USP13 and HA-MYC in HEK293T cells. D CHX chase assay (100 µg/ml) of control or FLAG-USP13 transfected HEK293T cells for indicated time points. β-actin is a loading control. E Immunoblot of USP13 and MYC in transfected HEK293T cells upon treatment with either DMSO or indicated concentrations of Spautin-1 for 24 h. F Schematic diagram showing the domain organization of USP13 with deletion constructs used. G Immunoblot analysis of HEK293T cells transfected with Myc-tagged WT or deletion mutants of USP13. Lysates were blotted by anti-Myc-tag and anti-MYC antibodies. Band intensities were quantified and normalized using β-actin levels. H Lysates from HEK293T cells expressing the indicated plasmids treated with MG132 (10 μM) for 6 hr were subjected to IP for FLAG-tagged MYC and then immunoblotting for HA-tagged ubiquitin. Total cell lysates (TCL) correspond to 10% of the total protein amount used for the precipitation. I The ∆Znf∆UBA mutant form of USP13 fails to deubiquitinate MYC. J USP13 knockdown (KD) increased the ubiquitin level of MYC. HEK293T cells (shNS, KD1, and KD2) were transfected with FLAG-MYC and HA-Ub. K HEK293T cells were transfected with FLAG-MYC, HA-Ub (WT, K48, or K63), and Myc-tagged USP13