FIGURE 9.
Distribution of Nrf2, Keap1, RNF4, and the PML-NB-marker protein SP100 in detergent-fractionated samples of HepG2 cells. HepG2 cells were grown in T-25 flasks. Some cultures were treated with As2O3 (2 μm) or MG-132 (10 μm). All cells were washed and scraped into ice-cold lysis buffer containing 1% Triton X-100. The lysate was centrifuged (14,000 × g) to separate DS fraction (supernatant) from DI fraction (pellet). The DI fraction was suspended in 500 μl of buffer containing equal volumes of Tris-HCl solution (pH 6.8) and 2× SDS sample buffer. All fractions were then resolved on an SDS-polyacrylamide gel (8%) and immunoblotted for SP100 (A), SUMO-1 (B), Keap1 (C), Nrf2 (D), and RNF4 (E). HMW, high molecular weight.