Figure 5.
PTPN23 bridges ESCRT-I and ESCRT-III, completing lysosomal degradation of tauRD (P301L). (A) Schematic view of WT PTPN23 and its mutants. (B) PTPN23 binds to UBAP1 via the CC domain. HEK293T cells were transfected with UBAP1-Myc, Flag-PTPN23, Flag-PTPN23 (ΔCC), and empty vector. Cells were lysed 48 h after transfection, and Flag-PTPN23 proteins were 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 two-tailed Student’s t test with ***P < 0.001. (C) PTPN23 binds to CHMP4B via residues L202 and I206 in the Bro1 domain. HEK293T cells were transfected with Venus-CHMP4B, Flag-PTPN23, Flag-PTPN23 (ΔBro1), Flag-PTPN23 (L202D/I206D), and empty vector. Cells were lysed 48 h after transfection, and Flag-PTPN23 proteins were immunoprecipitated. Input and IP samples were immunoblotted with the indicated antibodies. Data were normalized to the band intensity of Venus-CHMP4B in PTPN23 immunoprecipitated samples and represent the mean ± SEM (n = 3, from three independent experiments). N.D., not detected. (D) TauRD (P301L)-Venus aggregate–positive cells stably expressing mCherry (control) or mCherry-tagged sgRNA-resistant PTPN23 proteins illustrated in A were transfected with control or PTPN23 sgRNA, and successfully transfected cells were selected by puromycin selection. TauRD (P301L)-Venus fluorescence was measured by flow cytometer 1 wk after transfection. Data represent the Venus fluorescence from sgPTPN23-treated cells normalized against the Venus fluorescence from sgControl-treated cells. Data represent the mean ± SEM (n = 4, from four independent experiments). Significance was calculated using one-way ANOVA with Dunnett’s test with *P < 0.05. n.s., not significant. Source data are available for this figure: SourceData F5.

PTPN23 bridges ESCRT-I and ESCRT-III, completing lysosomal degradation of tauRD (P301L). (A) Schematic view of WT PTPN23 and its mutants. (B) PTPN23 binds to UBAP1 via the CC domain. HEK293T cells were transfected with UBAP1-Myc, Flag-PTPN23, Flag-PTPN23 (ΔCC), and empty vector. Cells were lysed 48 h after transfection, and Flag-PTPN23 proteins were 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 two-tailed Student’s t test with ***P < 0.001. (C) PTPN23 binds to CHMP4B via residues L202 and I206 in the Bro1 domain. HEK293T cells were transfected with Venus-CHMP4B, Flag-PTPN23, Flag-PTPN23 (ΔBro1), Flag-PTPN23 (L202D/I206D), and empty vector. Cells were lysed 48 h after transfection, and Flag-PTPN23 proteins were immunoprecipitated. Input and IP samples were immunoblotted with the indicated antibodies. Data were normalized to the band intensity of Venus-CHMP4B in PTPN23 immunoprecipitated samples and represent the mean ± SEM (n = 3, from three independent experiments). N.D., not detected. (D) TauRD (P301L)-Venus aggregate–positive cells stably expressing mCherry (control) or mCherry-tagged sgRNA-resistant PTPN23 proteins illustrated in A were transfected with control or PTPN23 sgRNA, and successfully transfected cells were selected by puromycin selection. TauRD (P301L)-Venus fluorescence was measured by flow cytometer 1 wk after transfection. Data represent the Venus fluorescence from sgPTPN23-treated cells normalized against the Venus fluorescence from sgControl-treated cells. Data represent the mean ± SEM (n = 4, from four independent experiments). Significance was calculated using one-way ANOVA with Dunnett’s test with *P < 0.05. n.s., not significant. Source data are available for this figure: SourceData F5.

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