CPT-induced Top1 down-regulation is dependent on the assembly of
K48-linked polyubiquitin chains.
a, HeLa cells were transfected
with the HA-Ub expression plasmid. Transfection, CPT treatment, and
immunoprecipitation (IP) were performed as described under
“Experimental Procedures.” b, HeLa cells were pretreated
with 10 μm APH or 150 μm DRB for 30 min, followed
by co-treatment with 25 μm CPT for 3 h. Top1 down-regulation was
analyzed as described under “Experimental Procedures.” c,
HeLa cells were transfected with various plasmids to overexpress HA-tagged
wild type and mutant ubiquitin proteins. Transfection, CPT treatment (25
μm), and Top1 down-regulation assay were performed as described
under “Experimental Procedures.” Ub, wild type ubiquitin;
K48R-Ub, K48R mutant ubiquitin; K29R-Ub, K29R mutant
ubiquitin; K63R-Ub, K63R mutant ubiquitin; K48-Ub, K48-only
ubiquitin (all lysine residues were mutated to arginine residues except
Lys-48); K63-only Ub (all lysine residues were mutated to arginine residues
except Lys-63). The amount of protein-HA-Ub conjugates in cells transfected
with each construct was also determined by immunoblotting using anti-HA
antibody (lower panels). d, Top1 degradation requires
ubiquitin and proteasome. HeLa cells were pretreated with 5 μm
MG132 or 5 μm G5 for 30 min, followed by co-treatment with 25
μm CPT for 3 h. Top1 down-regulation was determined by
immunoblotting of cell lysates with anti-hTop1 antibody. Cell lysates (from
control cells and G5-treated cells) were also immunoblotted with
anti-ubiquitin antibody to assess the levels of free ubiquitin (mono-Ub) and
ubiquitin-protein conjugates (poly-Ub). WB, Western blot;
DMSO, dimethyl sulfoxide.