Figure 5
Bucillamine induces the subcellular localization of Nrf2 in HEI-OC1 cells treated with cisplatin. (a) Cells grown in chamber slides were pretreated with 2 mM bucillamine for 1 h followed by the addition of 20 μM cisplatin for 12 h. Subcellular localization of Nrf2 was detected by indirect immunofluorescence staining with anti-Nrf2 antibodies followed by Alexa488–conjugated second antibodies (green). The nucleus (left panel) was stained with DAPI (blue), and fluorescent images were obtained. (b) Cytosolic (CE) and nuclear (NE) extracts from cells were separated on 12% SDS-PAGE and subjected to western blot analysis using anti-Nrf2 antibody. (c) Quantitative real-time PCR was carried out to determine the relative expression level of Nrf2 at 3 h. (d) Total RNA from cells was isolated with TRIzol reagent, cDNA was generated using reverse transcriptase, and PCR was performed to determine expression levels of Nrf2 at indicated time points.
