Figure 6.
HSP-associated UBAP1 disrupts tauRD (P301L) clearance. (A) Schematic view of WT UBAP1 and its mutants. (B) TauRD (P301L)-Venus aggregate–positive cells stably expressing mCherry (control) or mCherry-tagged sgRNA-resistant WT UBAP1 and its mutants illustrated in A were transfected with control or UBAP1 sgRNA, and successfully transfected cells were selected by puromycin. TauRD (P301L)-Venus fluorescence was measured by flow cytometer 1 wk after transfection. Data represent the Venus fluorescence from sgUBAP1-treated cells normalized against 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 Dunnett’s test with **P < 0.01, ***P < 0.001. n.s., not significant. (C) UBAP1 binds to PTPN23 via the PBR domain. HEK293T cells were transfected with Flag-PTPN23, UBAP1-Myc, UBAP1 (ΔSOUBA)-Myc, UBAP1 (ΔPBR)-Myc, and empty vector. Cells were lysed 48 h after transfection, and Flag-PTPN23 was immunoprecipitated. Input and IP samples were immunoblotted with the indicated antibodies. Data were normalized to the band intensity of UBAP1-Myc in PTPN23 immunoprecipitated samples and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Dunnett’s test with **P < 0.01. n.s. not significant. (D) Ring spot total intensity of tauRD (P301L)-Venus aggregates in cells stably expressing mCherry (Control) or mCherry-tagged WT UBAP1 and its mutants illustrated in A was quantified by a high-content image analyzer, and cells were imaged by a confocal microscope. Data were normalized to the ring spot total intensity of mCherry (control) cells and represent the mean ± SEM (n = 3). Significance was calculated using one-way ANOVA with Dunnett’s test with ***P < 0.001. n.s., not significant. Scale bar: 10 μm. Source data are available for this figure: SourceData F6.

HSP-associated UBAP1 disrupts tauRD (P301L) clearance. (A) Schematic view of WT UBAP1 and its mutants. (B) TauRD (P301L)-Venus aggregate–positive cells stably expressing mCherry (control) or mCherry-tagged sgRNA-resistant WT UBAP1 and its mutants illustrated in A were transfected with control or UBAP1 sgRNA, and successfully transfected cells were selected by puromycin. TauRD (P301L)-Venus fluorescence was measured by flow cytometer 1 wk after transfection. Data represent the Venus fluorescence from sgUBAP1-treated cells normalized against 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 Dunnett’s test with **P < 0.01, ***P < 0.001. n.s., not significant. (C) UBAP1 binds to PTPN23 via the PBR domain. HEK293T cells were transfected with Flag-PTPN23, UBAP1-Myc, UBAP1 (ΔSOUBA)-Myc, UBAP1 (ΔPBR)-Myc, and empty vector. Cells were lysed 48 h after transfection, and Flag-PTPN23 was immunoprecipitated. Input and IP samples were immunoblotted with the indicated antibodies. Data were normalized to the band intensity of UBAP1-Myc in PTPN23 immunoprecipitated samples and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Dunnett’s test with **P < 0.01. n.s. not significant. (D) Ring spot total intensity of tauRD (P301L)-Venus aggregates in cells stably expressing mCherry (Control) or mCherry-tagged WT UBAP1 and its mutants illustrated in A was quantified by a high-content image analyzer, and cells were imaged by a confocal microscope. Data were normalized to the ring spot total intensity of mCherry (control) cells and represent the mean ± SEM (n = 3). Significance was calculated using one-way ANOVA with Dunnett’s test with ***P < 0.001. n.s., not significant. Scale bar: 10 μm. Source data are available for this figure: SourceData F6.

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