USB1 ^P44L has an altered subcellular localization. (A–C) Western blot analysis of total cell (A) and cytosol vs. nucleus lysates (B and C) obtained from HEK293T cells ectopically expressing USB1-HA variants. Bars and error bars are averages of USB1-HA relative abundance normalized to HSP90 (cytosol) and SP1 (nucleus), and SD from four independent experiments. Two-way ANOVA statistical analysis was performed. “ns,” nonsignificant differences (P ≥ 0.05), *P < 0.05 (n = 4). (D) Semiautomatic quantification of immunofluorescence staining confirmed a decreased mean intensity nuclear vs. cytosolic ratio for HEK293T cells expressing USB1^P44L protein. Each independent experiment (n = 3) was represented with a different color. A minimum of 25 cells were analyzed for each independent experiment. An unpaired t test was performed. **P < 0.01. Source data are available for this figure: SourceData F2.