Arl8b depletion results in LAMP1 missorting to Rab11a-positive REs. (A) Representative confocal images of HeLa cells expressing SBP-GFP-LAMP1 (green) and stained for endogenous AP-3 (magenta) at the indicated time points after biotin addition. The line profiles indicate fluorescence intensity along the yellow lines for both channels: SBP-GFP-LAMP1 (green) and AP-3 (magenta). Scale bars: 10 μm (main); 2 μm (inset). (B) Manders’ colocalization coefficient quantification of SBP-GFP-LAMP1 with AP-3 at the indicated time points after biotin addition 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 = 0.0146; n.s., non-significant). The values are represented as the mean ± SEM. (C) Representative confocal micrographs of HeLa cells treated with control or Arl8b siRNA, followed by the expression of SBP-GFP-LAMP1 (green). Cells were fixed at 60 min after biotin addition and immunostained for endogenous AP-3 (magenta). The line profiles indicate fluorescence intensity along the yellow lines for both channels: SBP-GFP-LAMP1 (green) and AP-3 (magenta). Scale bars: 10 μm (main); 2 μm (inset). (D) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with AP-3 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 = 0.0088). The values are represented as the mean ± SEM. (E) Representative confocal images of HeLa cells treated with control or Arl8b siRNA, followed by the expression of SBP-GFP-LAMP1 (green). 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). (F) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with Rab11a or Rab14 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 = 0.0237; **P = 0.0028). The values are represented as the mean ± SEM. (G) HeLa cells stably expressing 3x-FLAG-TFR1 were treated with control or Arl8b siRNA, and the lysates were subjected to anti-FLAG IP. The precipitates were IB with the indicated antibodies. (H) Graph represents the relative fold change in densitometric values of the indicated proteins normalized to input and direct IP of 3x-FLAG-TFR1. Statistical significance was calculated using the one-sample t test (n = 4; *P = 0.0108; n.s., non-significant). The values are represented as the mean ± SEM. (I) Bar graph represents the percent MFI of surface LAMP1 in HeLa cells treated with the 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 = 0.0061; n.s., non-significant). The values are represented as the mean ± SEM. IP, immunoprecipitation; IB, immunoblotted. Source data are available for this figure: SourceData F4.