Figure 7.
TBC1D9B inactivates Rab11a for sorting of the newly synthesized LAMP1 to active lysosomes. (A) Representative confocal micrographs of HeLa cells treated with control or TBC1D9B siRNA and transfected with SBP-GFP-LAMP1. Cells were labeled with LTR (magenta) followed by biotin addition and imaged at the indicated time points. Scale bars: 10 µm (main); 2 μm (inset). (B) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with LTR in HeLa cells treated with indicated siRNAs is shown from three independent experiments (n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (****P < 0.0001). The values are represented as the mean ± SEM. (C) Representative confocal images of HeLa cells treated with control or TBC1D9B siRNA and expressing SBP-GFP-LAMP1. Cells were fixed at 60 min after biotin addition and immunostained for endogenous Rab11a (magenta). The line profiles indicate fluorescence intensity along the yellow lines for both channels: SBP-GFP-LAMP1 (green) and Rab11a (magenta). Scale bars: 10 μm (main); 2 μm (inset). (D) Manders’ colocalization coefficient quantification of SBP-GFP-LAMP1 with Rab11a in HeLa cells treated with the indicated siRNAs is shown from three independent experiments (n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (*P = 0.0168). The values are represented as the mean ± SEM. (E) HeLa cells stably expressing 3x-FLAG-TFR1 were treated with control or TBC1D9B siRNA, and lysates were subjected to anti-FLAG IP. The precipitates were IB with the indicated antibodies. (F) Bar graph represents the relative fold change in densitometric values of the indicated protein normalized to input and direct IP of 3x-FLAG-TFR1. Statistical significance was calculated using the one-sample t test (n = 4; **P = 0.0039; n.s., non-significant). The values are represented as the mean ± SEM. (G) Bar graph represents percent MFI of surface LAMP1 in HeLa cells treated with indicated siRNAs and normalized to control siRNA (n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (*P = 0.0275; n.s., non-significant). The values are represented as the mean ± SEM. Source data are available for this figure: SourceData F7. Refer to the image caption for details. Panel A shows confocal microscopy images of control or TBC1D9B siRNA-treated HeLa cells expressing SBP-GFP-LAMP1 and labeled with LysoTracker at multiple post-biotin incubation time points. Panel B shows scatter bar plot quantifying Manders colocalization coefficient between SBP-GFP-LAMP1 and LysoTracker in control or TBC1D9B siRNA-treated cells. Panel C shows confocal microscopy images and fluorescence intensity line profiles of control or TBC1D9B siRNA-treated HeLa cells expressing SBP-GFP-LAMP1 and immunostained for endogenous Rab11a. Panel D shows scatter bar plot quantifying Manders colocalization coefficient between SBP-GFP-LAMP1 and Rab11a in control or TBC1D9B siRNA-treated cells. Panel E shows immunoblot images of anti-FLAG immunoprecipitates from HeLa cells expressing 3x-FLAG-TFR1 treated with control or TBC1D9B siRNA and probed with indicated antibodies. Panel F shows a bar graph quantifying relative fold change in protein levels normalized to input and immunoprecipitation conditions. Panel G shows a bar graph comparing percentage mean fluorescence intensity of surface LAMP1 in HeLa cells treated with control siRNA, TBC1D9B siRNA, or combined TBC1D9B and Rab11a siRNA treatments.

TBC1D9B inactivates Rab11a for sorting of the newly synthesized LAMP1 to active lysosomes. (A) Representative confocal micrographs of HeLa cells treated with control or TBC1D9B siRNA and transfected with SBP-GFP-LAMP1. Cells were labeled with LTR (magenta) followed by biotin addition and imaged at the indicated time points. Scale bars: 10 µm (main); 2 μm (inset). (B) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with LTR in HeLa cells treated with indicated siRNAs is shown from three independent experiments (n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (****P < 0.0001). The values are represented as the mean ± SEM. (C) Representative confocal images of HeLa cells treated with control or TBC1D9B siRNA and expressing SBP-GFP-LAMP1. Cells were fixed at 60 min after biotin addition and immunostained for endogenous Rab11a (magenta). The line profiles indicate fluorescence intensity along the yellow lines for both channels: SBP-GFP-LAMP1 (green) and Rab11a (magenta). Scale bars: 10 μm (main); 2 μm (inset). (D) Manders’ colocalization coefficient quantification of SBP-GFP-LAMP1 with Rab11a in HeLa cells treated with the indicated siRNAs is shown from three independent experiments (n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (*P = 0.0168). The values are represented as the mean ± SEM. (E) HeLa cells stably expressing 3x-FLAG-TFR1 were treated with control or TBC1D9B siRNA, and lysates were subjected to anti-FLAG IP. The precipitates were IB with the indicated antibodies. (F) Bar graph represents the relative fold change in densitometric values of the indicated protein normalized to input and direct IP of 3x-FLAG-TFR1. Statistical significance was calculated using the one-sample t test (n = 4; **P = 0.0039; n.s., non-significant). The values are represented as the mean ± SEM. (G) Bar graph represents percent MFI of surface LAMP1 in HeLa cells treated with indicated siRNAs and normalized to control siRNA (n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (*P = 0.0275; n.s., non-significant). The values are represented as the mean ± SEM. Source data are available for this figure: SourceData F7.

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