Supplementary material to Fig. 2 . (A) HEK293A cells were transfected with control or TSG101 siRNA, and whole-cell lysates were immunoblotted with the indicated antibodies 72 h after transfection. (B) TauRD (P301L)-Venus aggregate–positive cells were transfected with control or TSG101 siRNA. Cells were lysed 72 h after transfection, and Triton X-100–soluble and Triton X-100–insoluble fractions and the conditioned medium were immunoprecipitated using a GST-tagged anti-GFP nanobody. Input and IP samples at the indicated percentages were immunoblotted with the indicated antibodies. (C) Single-cultured tauRD (P301L)-mCherry aggregate–negative cells, or cocultured tauRD (P301L)-mCherry aggregate–negative cells and tauRD (P301L)-Venus aggregate–positive cells were transfected with control or TSG101 siRNA. Cells were analyzed by flow cytometry 72 h after transfection. TauRD (P301L)-Venus fluorescence in tauRD (P301L)-mCherry cells was gated as R4. (D) Quantification of tauRD (P301L)-Venus fluorescence in R4. Data were normalized to the Venus fluorescence in siControl-treated cells and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using two-tailed Student’s t test with n.s., not significant. (E) Single-cultured or cocultured cells in C were sorted for tauRD (P301L)-mCherry–positive population by flow cytometry and immunoblotted with the indicated antibodies. Source data are available for this figure: SourceData FS3.