Fig. 3.

UBXN1 is required for the formation of aggresomes. (A) Immunoblot showing loss of UBXN1 in CRISPR/Cas9 generated knockout cells and re-expression of wild-type GFP–UBXN1 by doxycycline induction. (B) Wild-type (WT) and UBXN1 KO HeLa Flp-in TRex cell lines were treated with 1 µM Btz for 18 h. Cells were stained for ubiquitin. (C) Cell lysates from WT and KO cells treated with 1 µM Btz for 18 h were probed for ubiquitin. (D) GFP–UBXN1 expression in UBXN1 KO cells was induced by doxycycline. Cells were treated with Btz and stained for ubiquitin. Expression of GFP–UBXN1 reinstated aggresome formation in UBXN1 KO cells. (E) Quantification of data in B and D. (F) WT and UBXN1 KO lines were treated with 1 µM Btz for the indicated times and stained for ubiquitin. The black dot represents the mean from each biological replicate. The indicated number of cells (n) analyzed from all three independent biological replicates is shown for each condition. Graphs show the mean±s.e.m. *P≤0.05, **P≤0.01, as determined by one-way ANOVA with Bonferroni correction. Scale bars: 10 µm.