Fig. 2: NBR1 modulates p62–ubiquitin condensates formation in vitro.

Source data for the assays in this figure are provided as a Source Data file. a Schematic representation of the condensate formation assay. b Condensate formation assay with GST-4xUb (5 µM), mCherry-p62 (2 µM) and GFP-NBR1 (2 µM). Condensate formation over time in the mCherry and GFP channels was followed by spinning disk microscopy. Scale bar, 15 µm. c Quantification of the experiment in (b). An average of the number (left) and the size (right) of the condensates formed in the mCherry channel were measured with ImageJ and plotted with standard deviations against time for n = 3. The number of condensates formed in the GFP channel and quantification of the control experiment are shown in Supplementary Fig. 2a. A Coomassie stained SDS-Page gel of the proteins used for the assay is shown in Supplementary Fig. 2b. d Titration of GFP-NBR1 (concentration range: 0–2 µM) in a condensate formation assay containing 5 µM GST-4xUb and 2 µM mCherry-p62. The average number of mCherry-p62 condensates per field of imaging for n = 3 and standard deviations are plotted against time. Coomassie stained SDS-Page gel with protein inputs is shown in Supplementary Fig. 2c. e Quantification of a condensate formation assay performed with 5 µM GST-4xUb or in vitro synthesized K48- and K63-linked ubiquitin chains of various length (Supplementary Fig. 2d), incubated with mCherry-p62 (2 µM), with or without GFP-NBR1 (2 µM). BSA was added as a crowding agent to a final concentration of 2%. The average number of mCherry-p62 condensates for n = 3 and standard deviations are plotted against time. f Microscopy-based pull-down showing the recruitment of GFP-NBR1 (WT or ∆PB1 – 2 µM) to mCherry-p62 coated RFP-trap beads. Average GFP signal intensities and standard deviations for n = 3 are shown. An unpaired, two-tailed Student’s t test was used to estimate significance. P values are indicated in the figure. Purified NBR1 variants used for this experiment are shown in Supplementary Fig. 2e. g Microscopy-based pull-down showing the recruitment of GFP-NBR1 (WT or ∆UBA – 2 µM) to GST-4xUb coated glutathione beads. Average GFP signal intensities and standard deviations for n = 3 are shown. An unpaired, two-tailed Student’s t test was used to estimate significance. P values are indicated in the figure. Purified NBR1 variants used for this experiment are shown in Supplementary Fig. 2e. h Condensate formation assay was performed with NBR1 WT, ∆PB1, and ∆UBA variants (2 µM), mCherry-p62 (2 µM) and GST-4xUb (5 µM). The average number of mCherry-p62 condensates and standard deviations for n = 3 are plotted against time. Representative images of the GFP and mCherry channels at the 60 min time point are shown in Supplementary Fig. 2g. SDS-Page gels of purified recombinant GFP-NBR1 (WT and mutants) and the condensate formation assay protein inputs are shown in Supplementary Fig. 2e and f. i Condensate formation assay was performed with mCherry-p62 (2 µM) and GST-4xUb (5 µM) with the addition of GFP-NBR1 (2 µM) and/or its isolated PB1 domain (2 µM). The average number of mCherry-p62 condensates and standard deviations for n = 3 are plotted against time. SDS-Page gel with the protein inputs for the condensates assay is shown in Supplementary Fig. 2h.