Fig. 1

Smurf1 promotes the formation of p62-liquid droplets. A The Smurf1^+/+ and Smurf1^−/− MEF cells were transfected with RFP-p62, and fixed after treating with MG132 (20 µM, 12 h), and then immunofluorescence stained with anti-Smurf1 antibody. The nucleus was stained by DAPI. Bar: 5 µm. B The Smurf1^−/− MEF cells were transfected with RFP-p62 and fixed after transfection with HA or HA-Smurf1, and then immunofluorescence stained with anti-HA antibody. Bar: 5 µm. The nucleus was stained by DAPI. C, D LN229 cells treated with control or Smurf1 siRNA oligos were transfected with Flag or Flag-Smurf1-CS, fixed after treating with MG132 (20 µM, 12 h) and then performed by immunofluorescence analysis with anti-p62 antibody. The nucleus was stained with DAPI. Bar: 5 µm (C). Bar graphs indicate the number and size of 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 (D). E 293T cells lysates were immunoprecipitated by IgG or Smurf1 antibody. The immunoprecipitates and the input were probed with anti-p62 and anti-Smurf1 antibodies. F 293T cells were transfected with Flag, Flag-Smurf1 or Flag-Smurf1^C699A. Cell lysates were immunoprecipitated by anti-p62 antibody. The immunoprecipitates were probed with anti-Ub and anti-p62 antibodies. G, H 293T cells with GFP-p62 overexpression were transfected with Flag, Flag-Smurf1, Flag-Smurf1-C2, Flag-Smurf1-ΔC2, Flag-Smurf1-WW, Flag-Smurf1-ΔWW, Flag-Smurf1-HECT or Flag-Smurf1-ΔHECT (G, schematic diagram). Cell lysates were immunoprecipitated by anti-GFP antibody. The immunoprecipitates and the input were probed with anti-GFP and anti-Flag antibodies (H). I, J LN229 cells treated with control or Smurf1 siRNA oligos were transfected with Flag, Flag-Smurf1-CS, or Flag-Smurf1-CS-ΔWW, fixed after treating with MG132 (20 µM, 12 h) and then performed by immunofluorescence analysis with anti-p62 and anti-Flag antibody. The nucleus was stained with DAPI. Bar: 5 µm (I). Bar graphs indicate the number and size of 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 (J). K LN229 cells cultured in glass-bottom plates were transfected with GFP-p62 alone or co-transfected with GFP-Smurf1 or RFP-p62. Bar: 1 µm. The signal recovery after photobleaching was measured by Image J; mean ± SD, n = 20 droplets examined over three independent experiments. L LN229 cells with RFP or RFP-p62 overexpression were transfected with Flag or Flag-Smurf1, fixed, and then immunofluorescence stained with anti-Smurf1 antibody. The nucleus was stained with DAPI. Bar: 5 µm. The number of RFP-p62 puncta > 1 µm in each cell was assessed (mean ± SD, n = 10 cells examined over three independent experiments). * p < 0.05 as determined by unpaired two-tailed Student’s t-test. M LN229 cells treated with p62 siRNA oligos were transfected with Flag-Smurf1, then fixed after overexpressing RFP, RFP-p62, or RFP-p62^K7A/D69A. Bar: 5 µm. Bar graphs indicate the number and size of RFP-p62 puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). The nucleus was stained with DAPI. * p < 0.05, *** p < 0.001 as determined by unpaired two-tailed Student’s t-test