Fig. 2.

The p97 adaptors UBXN1 and NPL4 are localized to aggresomes. (A) HeLa Flp-in T-Rex cells were treated with 1 µM Btz for 18 h and cells were stained for UBXN1 and ubiquitin. Colocalization was determined through the Manders’ overlap coefficient for 25 cells in three replicate experiments. (B) NPL4, UFD1 and p47 localization to aggresomes labeled with ubiquitin in cells treated with 1 µM Btz for 18 h. Colocalization was determined through the Manders’ overlap coefficient for 25 cells in three replicate experiments. (C) GFP–UBXN1 colocalizes with the aggresome markers Proteostat, HDAC6 and 20S proteasomes in cells treated with 1 µM Btz for 18 h. (D) Microtubules are required for GFP–UBXN1 localization to aggresomes. Nocodazole co-incubation in Btz-treated (1 µM Btz for 18 h) cells prevents aggresome formation. Lower panel, the number of aggresomes was quantified. (E) Hela Flp-in T-Rex cells were treated with 0.1 mM sodium arsenite for 2 h. Cells were stained for stress granule marker G3BP1 and p97 adaptors (UBXN1 or p47, used here as a positive control. (F) Stable mCherry–Dcp1a cells (labeling P bodies) were stained with UBXN1. The indicated number of cells was analyzed from the three independent biological replicates. Manders’ coefficients are given as mean±s.e.m. For D, the box represents the tenth to 90th percentiles, and the median is indicated. The whiskers show the s.e.m. ***P≤0.001 as determined by unpaired Student's t-test. Scale bars: 10 µm.