Figure 4.
UEV domain of TSG101 is necessary for its recognition of ubiquitylated tauRD (P301L). (A) TauRD (P301L)-Venus aggregate–positive cells were treated with 100 nM TAK-243 for 24 h, and tauRD (P301L)-Venus fluorescence was measured by flow cytometry. Data were normalized to the Venus fluorescence of DMSO-treated cells and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using two-tailed Student’s t test with **P < 0.01. (B) Ring spot total intensity of tauRD (P301L)-Venus aggregates was quantified by a high-content image analyzer; cells were imaged by a confocal microscope 16 h after treatment of 100 nM TAK-243. Data were normalized to the ring spot total intensity of DMSO-treated cells and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using two-tailed Student’s t test with *P < 0.05. Scale bar: 10 μm. (C) Schematic view of the ubiquitin-binding domains of the ESCRT-I complex. (D) TauRD (P301L)-Venus aggregate–positive cells stably expressing mCherry (control) or mCherry-tagged sgRNA-resistant variants of WT UBAP1 and UBAP1 (ΔSOUBA) were transfected with control or UBAP1 sgRNA, and successfully transfected cells were selected by puromycin selection. TauRD (P301L)-Venus fluorescence was measured by flow cytometry 1 wk after transfection. Data represent the Venus fluorescence from sgUBAP1-treated cells normalized to the Venus fluorescence from sgControl-treated cells. Data represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Tukey’s test with *P < 0.05, **P < 0.01. n.s., not significant. (E) TauRD (P301L)-Venus aggregate–positive cells stably expressing mCherry (control) or mCherry-tagged siRNA-resistant variants of WT TSG101 and TSG101 (ΔUEV) were transfected with control or TSG101 siRNA. TauRD (P301L)-Venus fluorescence was measured by flow cytometry 72 h after transfection. Data represent the Venus fluorescence from siTSG101-treated cells normalized to the Venus fluorescence from siControl-treated cells. Data represent the mean ± SEM (n = 4, from four independent experiments). Significance was calculated using one-way ANOVA with Tukey’s test with *P < 0.05, **P < 0.01, ***P < 0.001. (F) TSG101 recognizes ubiquitylated proteins via the UEV domain. HEK293T cells were transfected with TSG101-Flag, TSG101 (ΔUEV)-Flag, and empty vector. Cells were lysed 48 h after transfection, and TSG101-Flag and TSG101 (ΔUEV)-Flag were immunoprecipitated. Input and IP samples were immunoblotted with the indicated antibodies. Data were normalized to the band intensity of ubiquitin in TSG101 immunoprecipitated samples and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using two-tailed Student’s t test with **P < 0.01. The asterisk denotes a nonspecific band. (G) Visualization of tauRD (P301L)-Venus aggregates and TSG101 and TSG101 (ΔUEV) in cells stably expressing mCherry-tagged WT TSG101 and TSG101 (ΔUEV). Endogenous TSG101 was depleted by siRNA knockdown. Images were acquired by a confocal microscope, and boxed areas are magnified at the top right corner. Pixel intensity correlation of tauRD (P301L)-Venus aggregates was calculated. Data represent the mean ± SEM (n = 60, from three independent experiments, 20 cells per experiment). Significance was calculated using two-tailed Student’s t test with ***P < 0.001. Scale bar: 10 and 5 μm (magnified images). (H) Visualization of tauRD (P301L)-Venus aggregates and TSG101 in tauRD (P301L)-Venus aggregate–positive cells stably expressing Tet-On mCherry-TSG101. After treating cells with 100 nM TAK-243 for 16 h, 1 μl/ml Dox was added, and cells were incubated for an additional 8 h. Images were acquired by a confocal microscope, and boxed areas are magnified at the top right corner. Pixel intensity correlation of tauRD (P301L)-Venus aggregates was calculated. Data represent the mean ± SEM (n = 60, from three independent experiments, 20 cells per experiment). Significance was calculated using two-tailed Student’s t test with ***P < 0.001. Scale bar: 10 and 5 μm (magnified images). Source data are available for this figure: SourceData F4.

UEV domain of TSG101 is necessary for its recognition of ubiquitylated tauRD (P301L). (A) TauRD (P301L)-Venus aggregate–positive cells were treated with 100 nM TAK-243 for 24 h, and tauRD (P301L)-Venus fluorescence was measured by flow cytometry. Data were normalized to the Venus fluorescence of DMSO-treated cells and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using two-tailed Student’s t test with **P < 0.01. (B) Ring spot total intensity of tauRD (P301L)-Venus aggregates was quantified by a high-content image analyzer; cells were imaged by a confocal microscope 16 h after treatment of 100 nM TAK-243. Data were normalized to the ring spot total intensity of DMSO-treated cells and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using two-tailed Student’s t test with *P < 0.05. Scale bar: 10 μm. (C) Schematic view of the ubiquitin-binding domains of the ESCRT-I complex. (D) TauRD (P301L)-Venus aggregate–positive cells stably expressing mCherry (control) or mCherry-tagged sgRNA-resistant variants of WT UBAP1 and UBAP1 (ΔSOUBA) were transfected with control or UBAP1 sgRNA, and successfully transfected cells were selected by puromycin selection. TauRD (P301L)-Venus fluorescence was measured by flow cytometry 1 wk after transfection. Data represent the Venus fluorescence from sgUBAP1-treated cells normalized to the Venus fluorescence from sgControl-treated cells. Data represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Tukey’s test with *P < 0.05, **P < 0.01. n.s., not significant. (E) TauRD (P301L)-Venus aggregate–positive cells stably expressing mCherry (control) or mCherry-tagged siRNA-resistant variants of WT TSG101 and TSG101 (ΔUEV) were transfected with control or TSG101 siRNA. TauRD (P301L)-Venus fluorescence was measured by flow cytometry 72 h after transfection. Data represent the Venus fluorescence from siTSG101-treated cells normalized to the Venus fluorescence from siControl-treated cells. Data represent the mean ± SEM (n = 4, from four independent experiments). Significance was calculated using one-way ANOVA with Tukey’s test with *P < 0.05, **P < 0.01, ***P < 0.001. (F) TSG101 recognizes ubiquitylated proteins via the UEV domain. HEK293T cells were transfected with TSG101-Flag, TSG101 (ΔUEV)-Flag, and empty vector. Cells were lysed 48 h after transfection, and TSG101-Flag and TSG101 (ΔUEV)-Flag were immunoprecipitated. Input and IP samples were immunoblotted with the indicated antibodies. Data were normalized to the band intensity of ubiquitin in TSG101 immunoprecipitated samples and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using two-tailed Student’s t test with **P < 0.01. The asterisk denotes a nonspecific band. (G) Visualization of tauRD (P301L)-Venus aggregates and TSG101 and TSG101 (ΔUEV) in cells stably expressing mCherry-tagged WT TSG101 and TSG101 (ΔUEV). Endogenous TSG101 was depleted by siRNA knockdown. Images were acquired by a confocal microscope, and boxed areas are magnified at the top right corner. Pixel intensity correlation of tauRD (P301L)-Venus aggregates was calculated. Data represent the mean ± SEM (n = 60, from three independent experiments, 20 cells per experiment). Significance was calculated using two-tailed Student’s t test with ***P < 0.001. Scale bar: 10 and 5 μm (magnified images). (H) Visualization of tauRD (P301L)-Venus aggregates and TSG101 in tauRD (P301L)-Venus aggregate–positive cells stably expressing Tet-On mCherry-TSG101. After treating cells with 100 nM TAK-243 for 16 h, 1 μl/ml Dox was added, and cells were incubated for an additional 8 h. Images were acquired by a confocal microscope, and boxed areas are magnified at the top right corner. Pixel intensity correlation of tauRD (P301L)-Venus aggregates was calculated. Data represent the mean ± SEM (n = 60, from three independent experiments, 20 cells per experiment). Significance was calculated using two-tailed Student’s t test with ***P < 0.001. Scale bar: 10 and 5 μm (magnified images). Source data are available for this figure: SourceData F4.

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