Fig. 4

Smurf1 induces the autophagic degradation of p62-liquid droplets. A LN229 cells were transfected with control or Smurf1 siRNA oligos, then treated with DMSO or BafA1 (100 nM, 24 h). Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-p62, anti-LC3B, anti-Smurf1, and anti-β-actin). Bar graphs indicate the quantitative densitometric analysis of LC3 relative to β-actin. Values were normalized against the intensity of LN229 transfected with si-Control and treated with DMSO; mean ± SD from 3 independent experiments. ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t-test. B LN229 cells were transfected with Flag or Flag-Smurf1, then treated with DMSO, BafA1 (100 nM, 24 h) or CQ (100 µM, 6 h). Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-Flag, anti-p62, anti-LC3B, and anti-β-actin). Bar graphs indicate the quantitative densitometric analysis of LC3 relative to β-actin. Values were normalized against the intensity of LN229 transfected with Flag and treated with DMSO; mean ± SD from 3 independent experiments. * p < 0.05, ** p < 0.01 as determined by unpaired two-tailed Student’s t-test. C LN229 cells with MG132 (20 µM, 12 h) treatment were fixed after treating with DMSO, EBSS (2 h), BafA1 (100 nM, 24 h), and then immunofluorescence stained with anti-p62, anti-Smurf1 and anti-LC3B antibodies. The nucleus was stained by DAPI. Bar: 5 µm. D 293T cells were treated with MG132. The lysates were immunoprecipitated by IgG or Smurf1 antibody. The immunoprecipitates and the input were probed with anti-LC3B and anti-Smurf1 antibodies. E The indicated His-Flag-tagged purified proteins of control and LC3 constructs were pulled down with Flag-beads separately, then incubated with GST-Smurf1. The proteins retained on beads were subjected to immunoblot using anti-GST and anti-Flag antibodies. F 293T cells were transfected with si-Control or si-p62 under MG132 treatment. The lysates were immunoprecipitated by IgG or Smurf1 antibody. The immunoprecipitates and the input were probed with anti-LC3B, anti-Smurf1 and anti-p62 antibodies. G LN229 cells treated by control or Smurf1 siRNA oligos were transfected with RFP-GFP-p62, then overexpressed Flag or Flag-Smurf1, and fixed after MG132 (20 µM, 12 h) treatment. Bar: 5 µm. The nucleus was stained by DAPI. Bar graphs indicate the number of RFP-GFP-p62 puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t-test