full length nbr1  (Addgene inc)

Journal: Cell & Bioscience

Article Title: Enhanced liquidity of p62 droplets mediated by Smurf1 links Nrf2 activation and autophagy

doi: 10.1186/s13578-023-00978-9

Figure Lengend Snippet: NBR1 enhances Smurf1-mediated p62 liquid-droplets accumulation. A , B LN229 cells were transfected with HA or HA-Smurf1. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-p62, anti-NBR1, anti-NDP52, anti-OPTN, anti-BAG3, anti-HA, and anti-β-actin) ( A ). Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to β-actin. Values were normalized against the intensity of LN229 transfected with HA; mean ± SD from 3 independent experiments. NS p > 0.05, *** p < 0.001 as determined by unpaired two-tailed Student’s t -test ( B ). C , D LN229 cells transfected with RFP-Smurf1 and GFP-p62 were treated with control or NBR1 siRNA oligos, fixed after 24 h transfection with HA or HA-NBR1. The nucleus was stained by DAPI. Bar: 5 µm ( C ). Bar graphs indicate the ratio of the number of RFP-Smurf + GFP-p62 + puncta to the number of GFP-p62 + puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). * p < 0.05 as determined by unpaired two-tailed Student’s t -test ( D ). E , F LN229 cells treated with control, NBR1, or Smurf1 siRNA oligos were transfected with GFP, GFP-Smurf1, or GFP-NBR1, fixed after treating with MG132 (20 µM, 12 h), and then immunofluorescence stained with anti-p62 antibody. Bar: 5 µm. The nucleus was stained by DAPI ( E ). Bar graphs indicate the number and size of p62 puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). NS p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t -test ( F ). G LN229 cells overexpressing Flag-Smurf1 were transfected with GFP or GFP-NBR1, fixed after treating with BafA1 (100 nM, 24 h) and/or MG132 (20 µM, 12 h), and then immunofluorescence stained with anti-p62 antibody. Bar graphs indicate the number and size of p62 puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). NS p > 0.05, * p < 0.05 as determined by unpaired two-tailed Student’s t -test. H , I LN229 cells transfected with RFP-GFP-p62 were treated by control, Smurf1 or NBR1 siRNA oligos, treated with MG132 (20 µM, 12 h), and fixed after transfection with HA, HA-NBR1 or Flag-Smurf1. The nucleus was stained by DAPI. Bar: 5 µm ( H ). Bar graphs indicate the number of RFP-GFP-p62 puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). NS p > 0.05, ** p < 0.01 as determined by unpaired two-tailed Student’s t -test ( I ). J LN229 cells transfected with GFP-p62 and Flag-Smurf1 cultured in glass-bottom plates were treated with control or NBR1 siRNA oligos, then overexpressed HA or HA-NBR1. The signal recovery after photobleaching was measured; mean ± SD, n = 20 droplets examined over three independent experiments. K LN229 cells transfected with GFP-p62 were treated with control or Smurf1 siRNA oligos, and overexpressed HA or HA-NBR1. The signal recovery after photobleaching was measured; mean ± SD, n = 20 droplets examined over three independent experiments

Article Snippet: Full-length NBR1 was amplified from pMXs-IP GFP-NBR1 (Addgene, #38283) and then inserted into PKH3-3 × HA and pEGFP-N3 vector.

Techniques: Transfection, Western Blot, Two Tailed Test, Control, Staining, Immunofluorescence, Cell Culture

Journal: Cell & Bioscience

Article Title: Enhanced liquidity of p62 droplets mediated by Smurf1 links Nrf2 activation and autophagy

doi: 10.1186/s13578-023-00978-9

Figure Lengend Snippet: 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)

Article Snippet: Full-length NBR1 was amplified from pMXs-IP GFP-NBR1 (Addgene, #38283) and then inserted into PKH3-3 × HA and pEGFP-N3 vector.

Techniques: Expressing, Control, Transfection, Western Blot, Immunoprecipitation, Two Tailed Test, Immunofluorescence, Staining

Journal: Cell & Bioscience

Article Title: Enhanced liquidity of p62 droplets mediated by Smurf1 links Nrf2 activation and autophagy

doi: 10.1186/s13578-023-00978-9

Figure Lengend Snippet: NBR1 enhances Smurf1-drived Nrf2-mediated oxidative stress response. A LN229 cells transfected with control, Nrf2 or p62 siRNA oligos were treated with control (PBS) or H 2 O 2 (200 µM, 2 h). Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-Smurf1, anti-NBR1, anti-p62, and anti-β-actin). B LN229 cells transfected with control, Nrf2 or p62 siRNA oligos were treated with control (PBS) or H 2 O 2 (200 µM, 2 h). Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of Smurf1 and NBR1 mRNA. Values were normalized against the amount of mRNA in LN229 treated with control siRNA oligos and control (PBS) (mean ± SD from 3 independent experiments). NS p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t -test. C LN229 cells were fixed after treating with control (PBS) or H 2 O 2 (200 µM, 2 h), and then immunofluorescence stained with anti-Smurf1, anti-p62, and anti-NBR1 antibodies. The nucleus was stained by DAPI. Bar: 5 µm. Bar graphs indicate the number and size of p62 puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). ** p < 0.01 as determined by unpaired two-tailed Student’s t -test. D LN229 cells were transfected with control or Smurf1 siRNA oligos. Thereafter, the cells were divided into three groups: (i) cultured in regular medium, (ii) treated with H 2 O 2 (200 µM, 2 h), and (iii) after treating with H 2 O 2 (200 µM, 2 h), cultured in regular medium for another 12 h. Cell lysates were prepared and subjected to western blot analysis with indicated antibodies (anti-NBR1, anti-p62, anti-p-p62 S349 , anti-p-p62 S403 , anti-Smurf1, and anti-β-actin). E LN229 cells transfected with control or Smurf1 siRNA oligos were treated with control (PBS) or H 2 O 2 (200 µM, 2 h). Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of Smurf1, p62, NBR1, and NQO1 mRNA. Values were normalized against the amount of mRNA in LN229 treated with control siRNA oligos and control (PBS) (mean ± SD from 3 independent experiments). NS p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t -test. F LN229 cells treated with control or Smurf1 siRNA oligos and fixed after treating with control (PBS) or H 2 O 2 (200 µM, 2 h), and then immunofluorescence stained with anti-NBR1, anti-Smurf1 and anti-p62 antibodies. The nucleus was stained by DAPI. Bar: 5 µm. 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. G LN229 cells transfected with control or p62 siRNA oligos were transfected with Flag or Flag-Smurf1, and treated with control (PBS) or H 2 O 2 (200 µM, 2 h) after overexpressing HA or HA-NBR1. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-p62, anti-NBR1, anti-Flag, and anti-β-actin). H LN229 cells transfected with control or p62 siRNA oligos were transfected with Flag or Flag-Smurf1, and treated with control (PBS) or H 2 O 2 (200 µM, 2 h) after overexpressing HA or HA-NBR1. Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of NQO1 mRNA. Values were normalized against the amount of mRNA in LN229 transfected with control siRNA oligos and Flag; mean ± SD from 3 independent experiments. NS p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t -test. I Role of Smurf1 in stress response. Stress upregulates the level of Smurf1, leading to the increased formation and material exchange of p62 liquid droplets. Smurf1 promotes the phosphorylation of p62 S349 by activating mTORC1 signalling pathway. These further promote the transcription of anti-stress proteins by competitively binding Keap1 and mediating Nrf2 nuclear import. The activated Nrf2 increases the mRNA level of Smurf1, p62, and NBR1 to promote the formation and enlarging of p62 liquid droplets as positive feedback

Article Snippet: Full-length NBR1 was amplified from pMXs-IP GFP-NBR1 (Addgene, #38283) and then inserted into PKH3-3 × HA and pEGFP-N3 vector.

Techniques: Transfection, Control, Western Blot, Two Tailed Test, Immunofluorescence, Staining, Cell Culture, Phospho-proteomics, Binding Assay