Figure 4
TFEB increases the stability of Nrf2. (a) The protein levels of TFEB and Nrf2 in H4 cells (Control) and H4 cell lines stably expressing TFEB (TFEB) were analyzed by immunoblotting using each corresponding antibody, respectively. (b) Dot plot represents the relative ratio of Nrf2 normalized with that of actin. (c) To examine the ubiquitination level of Nrf2 in H4 and TFEB cells, the cells were treated with 5 µM of MG132, a proteasome inhibitor, for 24 h. The cell lysates were used for the immunoprecipitation of Nrf2 using anti-Nrf2 antibody. The ubiquitination levels of Nrf2 were examined by immunoblotting using anti-ubiquitin antibody. (d) The cells were kept with or without 100 µM of cycloheximide (CHX) for 30 min. The protein levels of Nrf2 were analyzed by immunoblotting using anti-Nrf2 antibody. (e) Dot plot represents the relative ratio of Nrf2 in cells treated with CHX in contrast to those not treated. (f) HEK293 cells were transiently transfected with the Myc-Nrf2 expression plasmid and together with or without the pHM6-TFEB expression plasmid. The protein levels of Nrf2 and TFEB were analyzed by immunoblotting using anti-Myc and TFEB antibodies, respectively. The asterisk on the Nrf2 panel indicates a non-specific band. Full blots are provided in Supplementary Fig. S10. Data shown are mean ± S.E. of four independent experiments and were analyzed using Student’s t test. (*p < 0.05;  ***p < 0.001).
