Figure 2.

Regulation of TRUSS expression by Skp2. Huh-7 cells were transfected with expression vectors of HA-TRUSS (1 μg) along with 1.5 μg each of WT–Skp2, Flag-ΔF-Skp2 or ΔC Skp2 (A) or with increasing concentrations in combination with (0.5, 1 and 2 μg) of either Skp2 and Flag ΔF-Skp2 (1 μg) (B) or with WT–Skp2 and Flag-ΔF-Skp2 (C). Cell lysates were western-blotted for recombinant TRUSS, Skp2, c-Myc. Huh-7 cells were transiently transfected either with 2.5 μg WT-TRUSS alone or along with WT-Skp2 (D). After 48 h, cells were incubated with cycloheximide for indicated time points and the cell lysates were western-blotted for TRUSS, Skp2 and GAPDH. Huh-7 cells were metabolically labeled with [³⁵S] methionine/cysteine mix, pulse chased for indicated time periods flowed by the immunoprecipitation of cell lysates with anti-HA antibody resolved by SDS/PAGE and auto-radiographed (E). The total cell lysates were also western blotting for TRUSS and Skp2. GAPDH was used as internal control. (F) For ubiquitination assay, HEK293 cells were transfected with wild-type HA-TRUSS (1 μg), along with 2 μg each of His-Ub, WT-Skp2 and ΔF-Skp2 as indicated. The ubiquitinated proteins were pulled down under denaturing conditions using Ni-NTA agarose beads and were analyzed via protein gel blotting. The asterisk indicates a band that may correspond to TRUSS nonspecifically bound to the Ni-NTA beads.