Figure 3.
TBC1D20 regulates Rab11-mediated vesicle transport. (A) Peptide count from mass spectrometry analysis of TBC1D20-binding proteins was obtained from 293T cells stably expressing TurboID-GFP or TurboID-TBC1D20. (B) 293T cells were transfected with the indicated plasmids and subjected to immunoprecipitation. (C) GST pull-down showing the interaction between purified GST-Rab11a and purified His-TBC1D20 protein. (D and E) GAP activity about TBC1D20 toward Rab11. The GTPase activity of Rab11, catalyzed by TBC1D20, was measured by adding the proteins to a reaction solution in a 96-well plate. GTP with a final concentration of 400 μM was added to the reaction, and the absorbance at 360 nm was measured over time to assess enzymatic activity. Positive control, Evi5; Negative control, Rab7a. n = 3 independent experiments. (F and G) Immunofluorescence images (F) and quantification of the fluorescence intensity of Rab11a at the centrosome (G) for RPE1 GFP-Rab11a cells transfected with control or TBC1D20 siRNAs, cultured in serum medium, and stained with antibody against CEP164. n > 30 cells. (H and I) Immunofluorescence images (H) and quantification of the percentage of Rab11a at the centrosome (I) for RPE1 cells stably expressing WT TBC1D20, ResTBC1D20, or ResTBC1D20R105A, transfected with control or TBC1D20 siRNAs, and cultured in serum medium and stained with the antibodies against TBC1D20, CEP164, and Rab11. n > 30 cells. (J and K) Immunofluorescence images (J) and quantification of the fluorescence intensity of Rab11a at the centrosome (K) for RPE1 GFP-Rab11a cells transfected with HA-TBC1D20 or HA-TBC1D20R105A, cultured in serum medium, and stained with the antibody against HA-tag. n > 30 cells. (L and M) Immunofluorescence images (L) and quantification of the fluorescence intensity of Rab11b at the centrosome (M) for RPE1 GFP-Rab11B cells transfected with HA-TBC1D20 or HA-TBC1D20R105A, cultured in serum medium, and stained with the antibody against HA-tag. n > 30 cells. (N) Immunoprecipitation and immunoblotting showing the interaction between GFP-Rab11a or GFP-Rab11b and endogenous FIP2 in 293T cells. (O) Immunofluorescence images for RPE1 GFP-FIP2 cells transfected with HA-TBC1D20, cultured in serum medium, and stained with the antibody against HA-tag. The top row shows a cell untransfected with HA-TBC1D20, and the bottom row shows a cell transfected with HA-TBC1D20. All experiments were repeated at least three times. Data are presented as mean ± SEM. ***P < 0.001; ns, not significant. Scale bar, 5 µm. Source data are available for this figure: SourceData F3. Refer to the image caption for details.

TBC1D20 regulates Rab11-mediated vesicle transport. (A) Peptide count from mass spectrometry analysis of TBC1D20-binding proteins was obtained from 293T cells stably expressing TurboID-GFP or TurboID-TBC1D20. (B) 293T cells were transfected with the indicated plasmids and subjected to immunoprecipitation. (C) GST pull-down showing the interaction between purified GST-Rab11a and purified His-TBC1D20 protein. (D and E) GAP activity about TBC1D20 toward Rab11. The GTPase activity of Rab11, catalyzed by TBC1D20, was measured by adding the proteins to a reaction solution in a 96-well plate. GTP with a final concentration of 400 μM was added to the reaction, and the absorbance at 360 nm was measured over time to assess enzymatic activity. Positive control, Evi5; Negative control, Rab7a. n = 3 independent experiments. (F and G) Immunofluorescence images (F) and quantification of the fluorescence intensity of Rab11a at the centrosome (G) for RPE1 GFP-Rab11a cells transfected with control or TBC1D20 siRNAs, cultured in serum medium, and stained with antibody against CEP164. n > 30 cells. (H and I) Immunofluorescence images (H) and quantification of the percentage of Rab11a at the centrosome (I) for RPE1 cells stably expressing WT TBC1D20, ResTBC1D20, or ResTBC1D20R105A, transfected with control or TBC1D20 siRNAs, and cultured in serum medium and stained with the antibodies against TBC1D20, CEP164, and Rab11. n > 30 cells. (J and K) Immunofluorescence images (J) and quantification of the fluorescence intensity of Rab11a at the centrosome (K) for RPE1 GFP-Rab11a cells transfected with HA-TBC1D20 or HA-TBC1D20R105A, cultured in serum medium, and stained with the antibody against HA-tag. n > 30 cells. (L and M) Immunofluorescence images (L) and quantification of the fluorescence intensity of Rab11b at the centrosome (M) for RPE1 GFP-Rab11B cells transfected with HA-TBC1D20 or HA-TBC1D20R105A, cultured in serum medium, and stained with the antibody against HA-tag. n > 30 cells. (N) Immunoprecipitation and immunoblotting showing the interaction between GFP-Rab11a or GFP-Rab11b and endogenous FIP2 in 293T cells. (O) Immunofluorescence images for RPE1 GFP-FIP2 cells transfected with HA-TBC1D20, cultured in serum medium, and stained with the antibody against HA-tag. The top row shows a cell untransfected with HA-TBC1D20, and the bottom row shows a cell transfected with HA-TBC1D20. All experiments were repeated at least three times. Data are presented as mean ± SEM. ***P < 0.001; ns, not significant. Scale bar, 5 µm. Source data are available for this figure: SourceData F3.

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