Fig. 4
From: Nestin regulates cellular redox homeostasis in lung cancer through the Keap1–Nrf2 feedback loop
Nestin interferes with the Keap1-dependent ubiquitination of Nrf2 by competitively binding to Keap1. a qPCR analysis showing that knockdown of Nestin had no effect on Keap1 expression at the mRNA level. b Immunoblotting analysis showing that Nestin had no effect on Keap1 expression at the protein level. c Alteration of the Nestin levels had no influence on the ubiquitination of Keap1. Control or Nestin-knockdown cells transfected with or without a vector encoding Myc-Nestin were treated with 10 μM MG132 for 4 h and an in vivo ubiquitination assay was performed to determine the ubiquitination levels of Keap1. d Myc-Nestin plasmids were transfected into NSCLC cells, whole-cell lysates were immunoprecipitated with anti-Myc, and the precipitated proteins were blotted with the indicated antibodies. e Whole-cell lysates were immunoprecipitated with anti-Keap1 and the precipitated proteins were blotted with anti-Nestin, anti-Keap1, and anti-Nrf2. f The localizations of endogenous Keap1 and Nestin in NSCLC cells were determined by double-label indirect immunofluorescence with anti-Keap1 (red) and anti-Nestin (green) antibodies. The colocalization of Keap1 and Nestin is indicated by a yellow color in the merged images. Scale bar: 5 μm. g Nestin reduced the interaction between Nrf2 and Keap1. Control or Nestin-knockdown NSCLC cells were treated with 10 μM MG132 for 4 h. Cell lysates were immunoprecipitated with an anti-Keap1 antibody and blotted with an anti-Nrf2 antibody. N.S. represents no significant, Student’s t test. Source data are available as a Source Data file
