Fig. 6

Smurf1 mediated NBR1 expression in p62 dependent manner. A LN229 cells treated with control, p62 or NBR1 siRNA oligos were transfected with Flag or Flag-Smurf1. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-NBR1, anti-p62, and anti-β-actin). B 293T cells treated with control or p62 siRNA oligos were transfected with HA or HA-Smurf1, and immunoprecipitated by HA antibody. The immunoprecipitates and the input were probed with anti-p62, anti-NBR1, and anti-HA antibodies. C LN229 cells treated with control or p62 siRNA oligos were transfected with Flag or Flag-Smurf1. Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of NBR1 mRNA. Values were normalized against the amount of LN229 transfected with si-Control and Flag; mean ± SD from 3 independent experiments. * p < 0.05, ** p < 0.01 as determined by unpaired two-tailed Student’s t-test. D LN229 cells treated with control 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 (anti-Nrf2, anti-p62, anti-NBR1, anti-Smurf1 and anti-β-actin). E LN229 cells treated with control or Nrf2 siRNA oligos were transfected with Flag or Flag-Smurf1, fixed after treating with MG132 (20 µM, 12 h), and then immunofluorescence stained with anti-NBR1 and anti-p62 antibodies. The nucleus was stained by DAPI. Bar: 5 µm. Bar graphs indicate the number and size of NBR1⁺p62⁺ puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). *** p < 0.001 as determined by unpaired two-tailed Student’s t-test. F LN229 cells treated with control, Nrf2, or p62 siRNA oligos were transfected with HA or HA-NBR1, and then overexpressed Flag or Flag-Smurf1. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-p-p62^S349, anti-p62, anti-Flag, anti-HA, anti-Nrf2, and anti-β-actin)