Figure 4.
PAQR4 promotes Nrf2 protein nuclear localization through inhibiting Nrf2 degradation. (A-B) Total Nrf2 proteins were reduced by PAQR4 knockdown in indicated cells (A), which can be reversed by MG132 -proteasome inhibitor (B). Indicated total extracts were probed with Nrf2 and β-actin antibodies. (C) PAQR4 knockdown inhibits nuclear accumulation of Nrf2 proteins in SPC-A1 cells by western blot. Lamin B: nuclear fraction; GAPDH: cytosol fraction. (D) Relative mRNA expression of Nrf2 downstream targeted genes after PAQR4 knockdown by real-time RT-PCR in SPC-A1 cells. (E-G) Representative images of immunofluorescent staining of Nrf2 protein in indicated cell lines (E). (F) is the quantification data for (E) in SPC-A1 cells, (G) Quantification data for immunostaining of Nrf2 in GLC-82 cells. (H-I) Lysates of indicated cell lines treated by cycloheximide (CHX: 100 μg/mL) were examined by western blot with indicated antibodies. (I) Quantification data for (H). The dark arrow heads point to Nrf2 proteins based on previous findings 45. (J-M) Analysis of Nrf2 ubiquitination upon PAQR4 overexpression or knockdown was examined in indicated cells by western blot with differential antibodies: HA, Myc. Means±SEM, * P <0.05; ** P <0.01; *** P <0.001; t-test.