Fig. 3

Smurf1 increases the binding affinity between p62 and Keap1 to activate Nrf2. A LN229 cells were transfected with HA or HA-Smurf1. Cell lysates were subjected to western blot analysis with the indicated antibodies (anti-p62, anti-p-p62^S349, anti-p-p62^S403, anti-HA, and anti-β-actin). Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to β-actin (mean ± SD from 3 independent experiments). Values were normalized against the intensity of LN229 transfected with HA. * p < 0.05, ** p < 0.01 as determined by unpaired two-tailed Student’s t-test. B LN229 cells treated by p62 siRNA oligos were transfected with Flag or Flag-Smurf1, then overexpressed GFP, GFP-p62^WT, GFP-p62^S349A, or GFP-p62^K7A/D69A. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-p-p62^S349, anti-GFP, and anti-β-actin). C LN229 cells transfected with Flag or Flag-Smurf1 were treated with DMSO or Rapamycin (100 nM, 24 h). Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-p-p62^S349, anti-p62, anti-p-mTOR, anti-mTOR, anti-p-p70S6K, anti-p70S6K, anti-Flag and anti-β-actin). D LN229 cells treated by p62 siRNA oligos were transfected with Flag or Flag-Smurf1, then overexpressed GFP, GFP-p62^WT, GFP-p62^S349A or GFP-p62^K7A/D69A. Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of NQO1 mRNA. Values were normalized against the amount of NQO1 mRNA in LN229 transfected with GFP, Flag plasmids and p62 siRNA oligos; mean ± SD from 3 independent experiments. NS p > 0.05, * p < 0.05, *** p < 0.001 as determined by unpaired two-tailed Student’s t-test. E 293T cells overexpressed GFP-p62 were transfected HA, HA-Smurf1, si-Control, or si-Smurf1. Cell lysates were immunoprecipitated by anti-Keap1 antibody. The immunoprecipitates and the input were probed with anti-GFP, anti-Keap1, anti-Nrf2 and anti-HA antibodies. F 293T cells overexpressed GFP-p62^S349A were transfected HA, HA-Smurf1, or si-Smurf1. Cell lysates were immunoprecipitated by anti-Keap1 antibody. The immunoprecipitates and the input were probed with anti-GFP, anti-Keap1, anti-Nrf2 and anti-HA antibodies. G LN229 cells treated with control or Nrf2 siRNA oligos were transfected with Flag or Flag-Smurf1. Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of p62, NQO1, and HO1 mRNA. Values were normalized against the amount of mRNA in LN229 transfected with control siRNA oligos and Flag; mean ± SD from 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t-test. H LN229 cells treated with control, p62, or Nrf2 siRNA oligos were transfected with Flag or Flag-Smurf1. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies. I Smurf1 promotes the phosphorylation of p62^S349 by activating mTORC1 signalling pathway. p62^S349 phosphorylation enhance p62 binding affinity with Keap1 and Nrf2 nuclear translocation. The activated Nrf2 partially participates to the transcriptional activation of p62