Figure S2.
Arl8b localizes to the newly synthesized post-endocytic LAMP1 vesicles. (A) Lysates of WT and Arl8b-KI HeLa cells were IB with indicated antibodies. (B) Representative confocal micrographs from a live-cell imaging experiment of HeLa cells expressing the RUSH reporter (SBP-mCherry-LAMP1) and labeled with LTR. Scale bars: 10 μm (main); 2 μm (inset). (C) Quantification of Manders’ colocalization coefficient of SBP-mCherry-LAMP1 with LTR 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. (D) Representative images of HeLa cells co-expressing SBP-GFP-LAMP1 (green) and Arl8b-Halo (magenta), followed by live-cell imaging. Cells were incubated with biotin, and image insets for indicated time points are shown. The box with vertical lines represents blank space. Scale bars: 10 μm (main); 2 μm (inset). (E) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with Arl8b-Halo 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 by one-way ANOVA with Dunnett’s multiple comparisons test (*P = 0.0114; ***P = 0.0007; n.s., non-significant). The values are represented as the mean ± SEM. (F) Representative images of HeLa cells co-expressing SBP-GFP-LAMP1 (Y404A) (green) and Arl8b-Halo (magenta), followed by live-cell imaging. Cells were incubated with biotin, and image insets for indicated time points are shown. The black box with vertical lines represents blank space. Scale bars: 10 μm (main); 2 μm (inset). (G) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 (Y404A) with Arl8b-Halo at the indicated time points 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 by one-way ANOVA with Dunnett’s multiple comparisons test (n.s., non-significant). The values are represented as the mean ± SEM. (H) HeLa cell lysates treated with control or Vps33a siRNAs were IB for the indicated proteins. (I) HeLa cells treated with the control or Arl8b siRNA and expressing SBP-GFP-LAMP1 were imaged after 30 min of biotin addition along with pitstop2 (30 µM). The trafficking of newly synthesized RUSH-LAMP1 from the TGN to the plasma membrane was visualized by surface staining with an anti-GFP antibody, followed by labeling with Alexa Fluor 568–conjugated secondary antibody. Scale bar: 10 µm. (J) Graph represents the relative ratio of surface signal (measured by an anti-GFP antibody) to total GFP signal of SBP-GFP-LAMP1 in HeLa cells treated with indicated siRNAs (n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (n.s., non-significant). The values are represented as the mean ± SEM. Source data are available for this figure: SourceData FS2. Refer to the image caption for details. Panel A shows immunoblot images comparing protein expression in wild-type and Arl8b knock-in HeLa cell lysates using indicated antibodies. Panel B shows live-cell confocal microscopy images of HeLa cells expressing SBP-mCherry-LAMP1 and labeled with LysoTracker at different time points following biotin addition. Panel C shows scatter bar plots quantifying Manders colocalization coefficient between SBP-mCherry-LAMP1 and LysoTracker from multiple independent live-cell imaging experiments. Panel D shows live-cell microscopy images and fluorescence intensity line profiles of HeLa cells co-expressing SBP-GFP-LAMP1 and Arl8b-Halo at different post-biotin incubation time points. Panel E shows scatter bar plots quantifying Manders colocalization coefficient between SBP-GFP-LAMP1 and Arl8b-Halo across multiple post-biotin incubation time points. Panel F shows live-cell microscopy images and fluorescence intensity line profiles of HeLa cells co-expressing SBP-GFP-LAMP1 Y404A and Arl8b-Halo at different post-biotin incubation time points. Panel G shows scatter bar plots quantifying Manders colocalization coefficient between SBP-GFP-LAMP1 Y404A and Arl8b-Halo across multiple post-biotin incubation time points. Panel H shows immunoblot images of HeLa cell lysates treated with control or Vps33a siRNA and probed with indicated antibodies. Panel I shows fluorescence microscopy images of HeLa cells treated with control or Arl8b siRNA expressing SBP-GFP-LAMP1 following biotin addition and surface anti-GFP antibody staining. Panel J shows a scatter bar plot quantifying the relative ratio of surface anti-GFP signal to total SBP-GFP-LAMP1 signal in HeLa cells treated with indicated siRNAs.

Arl8b localizes to the newly synthesized post-endocytic LAMP1 vesicles. (A) Lysates of WT and Arl8b-KI HeLa cells were IB with indicated antibodies. (B) Representative confocal micrographs from a live-cell imaging experiment of HeLa cells expressing the RUSH reporter (SBP-mCherry-LAMP1) and labeled with LTR. Scale bars: 10 μm (main); 2 μm (inset). (C) Quantification of Manders’ colocalization coefficient of SBP-mCherry-LAMP1 with LTR 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. (D) Representative images of HeLa cells co-expressing SBP-GFP-LAMP1 (green) and Arl8b-Halo (magenta), followed by live-cell imaging. Cells were incubated with biotin, and image insets for indicated time points are shown. The box with vertical lines represents blank space. Scale bars: 10 μm (main); 2 μm (inset). (E) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with Arl8b-Halo 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 by one-way ANOVA with Dunnett’s multiple comparisons test (*P = 0.0114; ***P = 0.0007; n.s., non-significant). The values are represented as the mean ± SEM. (F) Representative images of HeLa cells co-expressing SBP-GFP-LAMP1 (Y404A) (green) and Arl8b-Halo (magenta), followed by live-cell imaging. Cells were incubated with biotin, and image insets for indicated time points are shown. The black box with vertical lines represents blank space. Scale bars: 10 μm (main); 2 μm (inset). (G) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 (Y404A) with Arl8b-Halo at the indicated time points 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 by one-way ANOVA with Dunnett’s multiple comparisons test (n.s., non-significant). The values are represented as the mean ± SEM. (H) HeLa cell lysates treated with control or Vps33a siRNAs were IB for the indicated proteins. (I) HeLa cells treated with the control or Arl8b siRNA and expressing SBP-GFP-LAMP1 were imaged after 30 min of biotin addition along with pitstop2 (30 µM). The trafficking of newly synthesized RUSH-LAMP1 from the TGN to the plasma membrane was visualized by surface staining with an anti-GFP antibody, followed by labeling with Alexa Fluor 568–conjugated secondary antibody. Scale bar: 10 µm. (J) Graph represents the relative ratio of surface signal (measured by an anti-GFP antibody) to total GFP signal of SBP-GFP-LAMP1 in HeLa cells treated with indicated siRNAs (n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (n.s., non-significant). The values are represented as the mean ± SEM. Source data are available for this figure: SourceData FS2.

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