Figure 6.
TBC1D9B localizes on peripheral non-acidic LAMP1-positive vesicles in an Arl8b-dependent manner. (A) Representative confocal micrographs of HeLa cells co-transfected with GFP-TBC1D9B (WT, E91A, or 1–270 aa) with Arl8b (WT, Q75L, or T34N)-Halo, followed by live-cell imaging. Micrographs are maximum-intensity projections of z-stack images from live-cell video. The line profiles indicate fluorescence intensity of GFP-TBC1D9B (green) and Arl8b-Halo (magenta) along the yellow line. Scale bars: 10 µm (main); 2 µm (inset). (B) Quantification of PCC of GFP-TBC1D9B with Arl8b-Halo is shown from three independent experiments (n = 3). Colors in the SuperPlots indicate GFP-TBC1D9B (WT, E91A, or 1–270 aa), with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparisons test (****P < 0.0001). The values are represented as the mean ± SEM. (C) Quantification of Manders’ colocalization coefficient of GFP-TBC1D9B with Arl8b-Halo is shown from three independent experiments (n = 3). Colors in the SuperPlots indicate GFP-TBC1D9B (WT, E91A, or 1–270 aa), with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparisons test (****P < 0.0001). The values are represented as the mean ± SEM. (D) Representative images of HeLa cells co-expressing GFP-TBC1D9B and LAMP1-RFP (upper image)/LTR (lower image) with Arl8b-HA (not shown). The line profiles indicate fluorescence intensity of GFP-TBC1D9B (green) and LAMP1-RFP or LTR (magenta) along the yellow line. Scale bars: 10 µm (main); 2 µm (inset). (E) Quantification of Manders’ colocalization coefficient of GFP-TBC1D9B with indicated markers 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. The values are represented as the mean ± SEM. (F) HEK293T cells stably expressing TMEM192-2x-FLAG were treated with control or Arl8b siRNA, and lysates were subjected to anti-FLAG IP. The precipitates were IB with the indicated antibodies. (G) Bar graph represents the relative fold change in densitometric values of the indicated protein normalized to input and direct IP of TMEM192-2x-FLAG. Statistical significance was calculated using the one-sample t test (n = 3; **P = 0.0054; n.s., non-significant). The values are represented as the mean ± SEM. (H) Representative time-lapse confocal micrograph of a HeLa cell, co-expressing GFP-TBC1D9B (green), RUFY1-Halo (yellow), and Arl8b-DsRed (magenta). Micrographs are maximum-intensity projections of z-stack images from a live-cell video. The line profiles indicate fluorescence intensity along the blue lines for all channels: GFP-TBC1D9B (green), RUFY1-Halo (yellow), and Arl8b-DsRed (magenta). Scale bars: 10 µm (main); 2 µm (inset). Source data are available for this figure: SourceData F6. Refer to the image caption for details. Panel A shows confocal microscopy images and fluorescence intensity line profiles of HeLa cells co-expressing GFP-TBC1D9B variants with Arl8b Halo constructs, highlighting their colocalization patterns at different conditions. Panel B shows scatter bar plots quantifying Pearsons correlation coefficient between GFP-TBC1D9B variants and Arl8b Halo across multiple experimental conditions. Panel C shows scatter bar plots quantifying Manders colocalization coefficient between GFP-TBC1D9B variants and Arl8b Halo across multiple experimental conditions. Panel D shows confocal microscopy images and fluorescence intensity line profiles of GFP-TBC1D9B with LAMP1-RFP or LysoTracker in the presence of Arl8b. Panel E shows scatter bar plots quantifying Manders colocalization coefficient of GFP-TBC1D9B with LAMP1-RFP and LysoTracker. Panel F shows immunoblot images of anti-FLAG immunoprecipitates from HEK293T cells expressing TMEM192-2x-FLAG treated with control or Arl8b siRNA. Panel G shows a bar graph quantifying relative fold change in protein levels normalized to input and immunoprecipitation conditions. Panel H shows time-lapse confocal microscopy images and fluorescence intensity line profiles of HeLa cells co-expressing GFP-TBC1D9B, RUFY1-Halo, and Arl8b-DsRed, highlighting dynamic vesicle interactions.

TBC1D9B localizes on peripheral non-acidic LAMP1-positive vesicles in an Arl8b-dependent manner. (A) Representative confocal micrographs of HeLa cells co-transfected with GFP-TBC1D9B (WT, E91A, or 1–270 aa) with Arl8b (WT, Q75L, or T34N)-Halo, followed by live-cell imaging. Micrographs are maximum-intensity projections of z-stack images from live-cell video. The line profiles indicate fluorescence intensity of GFP-TBC1D9B (green) and Arl8b-Halo (magenta) along the yellow line. Scale bars: 10 µm (main); 2 µm (inset). (B) Quantification of PCC of GFP-TBC1D9B with Arl8b-Halo is shown from three independent experiments (n = 3). Colors in the SuperPlots indicate GFP-TBC1D9B (WT, E91A, or 1–270 aa), with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparisons test (****P < 0.0001). The values are represented as the mean ± SEM. (C) Quantification of Manders’ colocalization coefficient of GFP-TBC1D9B with Arl8b-Halo is shown from three independent experiments (n = 3). Colors in the SuperPlots indicate GFP-TBC1D9B (WT, E91A, or 1–270 aa), with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparisons test (****P < 0.0001). The values are represented as the mean ± SEM. (D) Representative images of HeLa cells co-expressing GFP-TBC1D9B and LAMP1-RFP (upper image)/LTR (lower image) with Arl8b-HA (not shown). The line profiles indicate fluorescence intensity of GFP-TBC1D9B (green) and LAMP1-RFP or LTR (magenta) along the yellow line. Scale bars: 10 µm (main); 2 µm (inset). (E) Quantification of Manders’ colocalization coefficient of GFP-TBC1D9B with indicated markers 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. The values are represented as the mean ± SEM. (F) HEK293T cells stably expressing TMEM192-2x-FLAG were treated with control or Arl8b siRNA, and lysates were subjected to anti-FLAG IP. The precipitates were IB with the indicated antibodies. (G) Bar graph represents the relative fold change in densitometric values of the indicated protein normalized to input and direct IP of TMEM192-2x-FLAG. Statistical significance was calculated using the one-sample t test (n = 3; **P = 0.0054; n.s., non-significant). The values are represented as the mean ± SEM. (H) Representative time-lapse confocal micrograph of a HeLa cell, co-expressing GFP-TBC1D9B (green), RUFY1-Halo (yellow), and Arl8b-DsRed (magenta). Micrographs are maximum-intensity projections of z-stack images from a live-cell video. The line profiles indicate fluorescence intensity along the blue lines for all channels: GFP-TBC1D9B (green), RUFY1-Halo (yellow), and Arl8b-DsRed (magenta). Scale bars: 10 µm (main); 2 µm (inset). Source data are available for this figure: SourceData F6.

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