FIG. 5.

PIAS-1 stabilizes the p73 protein. (A) H1299 cells were transfected with HA-TAp73α alone or with increasing amounts of Flag-PIAS-1, and all samples were also cotransfected with equal amounts of a GFP-expressing plasmid. At 24 h after transfection, cell extracts were lysed andthe levels of p73 in whole-cell extracts were determined by immunoblotting (IB) with anti-HA antibody (upper panel). The same blot was reprobed with anti-Flag antibody (second panel) and with an antiactin antibody (lower panel). To demonstrate that there is no effect of PIAS-1 on unrelated proteins or on the promoter driving expression of p73, the blot was reprobed with anti-GFP antibody. (B) ΔNp73 is also stabilized by PIAS-1 coexpression. H1299 cells were transfected with either HA-TAp73α or HA-ΔNp73α together with PIAS-1. At 24 h after transfection, cell extracts were lysed and analyzed as described for panel A. (C) ³⁵S pulse-chase. H1299 cells were transfected with the indicated plasmids. At 48 h posttransfection cells were labeled with l-³⁵S in vitro cell labeling mix. Cells were collected at the indicated time points. Immunoprecipitation (IP) was performed with anti-HA (Y-11) polyclonal antibody (Santa Cruz), and then samples were run on a polyacrylamide gel and proteins were detected by autoradiography. (D) The relative amount of p73 protein was evaluated by densitometry. (E) Evaluation of TAp73 protein half-life. Cycloheximide was added to H1299 cells at 24 h after transfection with the indicated plasmids. p73 or tubulin protein levels were determined by collecting cells at the indicated time points and performing immunoblotting as described above. (F) The relative amount of p73 protein was evaluated by densitometry and normalized to tubulin.