Multiple Interactions between Peroxisome Proliferators-Activated Receptors and the Ubiquitin-Proteasome System and Implications for Cancer Pathogenesis
Figure 2
Ubiquitination of truncated and
mutated forms of PPAR. (a) U2OS cells
were transfected with HA-ubiquitin expressing vector along with wild type
His-PPAR or truncated forms of the receptor (PPAR/Tr1-204, Tr1-235 and Tr1-299). After 24 hours,
cells were incubated overnight with vehicle or the PPAR ligand GW501516 (5 μM) and subsequently all samples were incubated with
10 μM the proteasome inhibitor PS341 for 4 hours.
His-tagged wild type and truncated PPAR were pulled-down with nickel affinity gel
under denaturing conditions. PPAR was detected in pull-down fractions using an
anti-His antibody and ubiquitinated proteins with an anti-HA antibody. (b) U2OS
cells were transfected with HA-ubiquitin vector along with the indicated PPAR expressing vectors. PPAR/Tr1-235 had wild type sequence or the
indicated double or triple mutations (K204R, K224R and K229R). Cells were
treated and analyzed as above.
