Fig. 4.

JAB1 represses transactivational activity of Luman and promotes degradation of the Luman protein. (A) Dual luciferase reporter assays. HEK-293 cells were transiently transfected with pHA-JAB1 and FLAG-Luman (top panel) or Gal DBD-Luman (bottom panel) constructs, along with the firefly luciferase reporter plasmids 5×UPRE or 5×GAL4 respectively. The control Renilla luciferase reporter plasmid pRL-SV40 was included in all samples. The firefly luciferase values are normalized to Renilla luciferase before being referenced to the pcDNA control. Data are based on 3 independent assays and shown with standard errors. (B) Pulse-chase assays. HEK-293 cells were transiently transfected with pcFLAG-Luman alone, or co-transfected with HA-JAB1. The cells were methionine/cysteine starved for 1 h, pulse-labeled with [³⁵S]-methionine/cysteine for 1.5 h and chased for the time as indicated. The Luman protein was then immunoprecipitated with an anti-FLAG antibody, resolved by SDS-PAGE, and visualized by autoradiography. The negative control represents a pooled sample immunoprecipitated with a non-specific antibody. The half-life of the protein was determined by densitometric analysis using data from two independent trials collected on a Typhoon 9400 Phosphorimager. Values are plotted with standard errors.