Figure S4. Supplementary material to... | Rockefeller University Press

Figure S4.

$Supplementary material toFigs. 3 and 4. (A and B) K48 ubiquitin tetramer (A) and K63 ubiquitin tetramer (B) were treated with OTUB1 or AMSH-LP at the indicated concentrations and rotated at 37°C for 30 min and then analyzed on an SDS-PAGE gel or immunoblotted with the indicated antibodies. (C) 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. Whole-cell lysates were immunoblotted with the indicated antibodies 1 wk after transfection. (D) 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. Whole-cell lysates were immunoblotted with the indicated antibodies 72 h after transfection. (E) Visualization of tauRD (P301L)-Venus aggregates and UBAP1 and UBAP1 (ΔSOUBA) in cells stably expressing mCherry-tagged WT UBAP1 and UBAP1 (ΔSOUBA). Endogenous UBAP1 was depleted by sgRNA KO. 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 < 0.05. Scale bar: 10 and 5 μm (magnified images). (F) TauRD (P301L)-Venus aggregate–positive cells stably expressing Tet-on tauRD (P301L/all-KR)-HA-P2A-iRFP670 aggregate–positive cells were transfected with control or TSG101 siRNA. 1 μl/ml Dox was added at the time of transfection. Cells were lysed 72 h after transfection, and Triton X-100–soluble and Triton X-100–insoluble fractions were immunoprecipitated with a HA antibody. Input and IP samples were immunoblotted with the indicated antibodies with S, soluble, and I, insoluble. The asterisk denotes a nonspecific band. A double dagger denotes the light chain of Protein G. Source data are available for this figure: SourceData FS4.$

Supplementary material to Figs. 3 and 4. (A and B) K48 ubiquitin tetramer (A) and K63 ubiquitin tetramer (B) were treated with OTUB1 or AMSH-LP at the indicated concentrations and rotated at 37°C for 30 min and then analyzed on an SDS-PAGE gel or immunoblotted with the indicated antibodies. (C) 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. Whole-cell lysates were immunoblotted with the indicated antibodies 1 wk after transfection. (D) 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. Whole-cell lysates were immunoblotted with the indicated antibodies 72 h after transfection. (E) Visualization of tauRD (P301L)-Venus aggregates and UBAP1 and UBAP1 (ΔSOUBA) in cells stably expressing mCherry-tagged WT UBAP1 and UBAP1 (ΔSOUBA). Endogenous UBAP1 was depleted by sgRNA KO. 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 < 0.05. Scale bar: 10 and 5 μm (magnified images). (F) TauRD (P301L)-Venus aggregate–positive cells stably expressing Tet-on tauRD (P301L/all-KR)-HA-P2A-iRFP670 aggregate–positive cells were transfected with control or TSG101 siRNA. 1 μl/ml Dox was added at the time of transfection. Cells were lysed 72 h after transfection, and Triton X-100–soluble and Triton X-100–insoluble fractions were immunoprecipitated with a HA antibody. Input and IP samples were immunoblotted with the indicated antibodies with S, soluble, and I, insoluble. The asterisk denotes a nonspecific band. A double dagger denotes the light chain of Protein G. Source data are available for this figure: SourceData FS4.

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