FIGURE 9.

Effect of PTMα siRNA on ubiquitination and degradation of cytosolic and nuclear Nrf2. A, Hepa-1 cells were transfected with 75 nm control or PTMα siRNA for 10 h and then co-transfected with HA-ubiquitin (UB), FLAG-INrf2, and Nrf2-V5 plasmid DNA for 36 h. The cells were harvested, and cytosolic and nuclear extracts were prepared. 1 mg of extracts was immunoprecipitated with anti-V5 antibodies and Western-blotted (WB) with HA-HRP tag antibody (ab, upper panel). Densitometry of the Nrf2 ubiquitinated bands were plotted (lower panel). Error bars indicate S.E. of triplicate samples. Statistical analysis was performed by one way analysis of variance followed by the Tukey-Kramer's post hoc test for multiple comparisons. ^*, p < 0.003 (compared with ubiquitinated nuclear Nrf2 of cells transfected with mock or control siRNA). B, 60 μg of cytosolic and nuclear extracts in the above experiment were immunoblotted with anti-FLAG-HRP and anti-V5-HRP antibodies. The same extracts were also probed with anti-PTMα, anti-LDH, and anti-lamin B antibodies for detection of endogenous proteins.