The occurrence of a double-crossover ΔpknG mutant was confirmed by its inability to grow after 1 day at 30°C on MM agar containing 25 μg mL−1 kanamycin.
sipD null mutant was confirmed by colony PCR.
The mutant was confirmed by restriction digestions and by DNA sequencing.
The final mutant was confirmed by PCR of the hemO region.
The bacterial accumulation of each isolated mutant was confirmed using the fluorescence stereomicroscope.
The expression of optineurin and its mutant was confirmed by western blotting (Figure 3A).
The absence of the mamK gene from the mutant was confirmed by PCR analysis (data not shown).
Abolishment of Nrf2 phosphorylation in the Nrf2-5A mutant was confirmed by the in vivo phosphorylation assay (Figure 3A).
Each single mutant was confirmed by sequencing of the cDNA and was then cloned into the pCIneo (Promega) mammalian expression vector.
The defective transcription activation of this mutant was confirmed and extended to other Adr1-dependent genes by measuring RNA levels with quantitative real-time PCR (Figure 5).
