Figure 2. Non-cleavable Nrf1 mutant is functionally defective.
(A) Nrf1−/− MEFs were transfected with constructs expressing either Nrf13×Flag, Nrf1(m1)3×Flag, or vector control and 48 hr later were subjected to Geneticin selection. These antibiotic resistant cells were treated with 1 µM MG132 for 10 hr as indicated. Lysates from these cells were immunoblotted with anti-Flag antibodies to detect exogenous Nrf1. β-actin was used as a loading control. (B) RNA from Nrf1−/− MEFs transfected and selected as described above was used for quantitative RT-PCR to assess the mRNA levels of representative PSM genes. The values were normalized to GAPDH mRNA levels. Error bars denote SD (n = 3).