Fig 2: Effects of Rab small GTPases on the peripheral localisation of CD68. (a) Expression of various Rab proteins in osteoclasts. Splenic macrophages from wild-type mice were infected with a retrovirus carrying a gene encoding FLAG-tagged Rab7 (left panels), Rab11B (middle panels) or Rab27A (right panels) and cultured with RANKL for 6 days. Then, osteoclast lysates were electrophoresed through an acrylamide gel. FLAG-Rab proteins were detected with antibodies specific for FLAG (upper panel, magenta arrows) and each Rab protein (middle panel, magenta arrows). Endogenous Rab proteins (middle panel, closed arrows) and ß-actin (lower panel) were also detected using corresponding antibodies. WT, DN and CA indicate cells expressing wild-type, dominant-negative (GDP-bound) and constitutively active (GTP-bound) Rab proteins, respectively. Control indicates cells infected with an empty vector. Bars indicate the position of the 25 kDa molecular mass marker. Numbers below blots represent relative signal intensities of FLAG-tagged (magenta) and endogenous (black) Rab protein normalised to that of FLAG-tagged wild-type Rab protein. Unprocessed scans of immunoblots are shown in Supplementary Fig. S6. (b) Effects of Rab protein expression on the peripheral localisation of CD68. Osteoclasts expressing Rab proteins were prepared as described in (a) and stained with antibodies against FLAG (green), CD68 (red) and a-tubulin (white). Merged images (Merge) are also shown. a-tubulin labelling is shown in blue in the merged images. WT, DN, CA and Control are as described in (a). The images are representative of at least nine cells. The bar indicates 20 µm. (c) Distribution of CD68 in osteoclasts expressing dominant-negative Rab proteins. Osteoclasts were stained as described in (b). The intensity of CD68 staining in each section was quantified and summarised as described in Fig. 3e. Blue, orange, green and purple indicate control and dominant-negative Rab7-, Rab11B- and Rab27A-expressing osteoclasts, respectively. Data are means ± s.e.m.; n = 15 cells.

Fig 3: IN1 treatment prevents recycling cargo sorting into the Rab11a-dependent pathway of the recycling circuit in MCMV infected cells. Cells infected with ?m138-MCMV were treated with 3 µm IN1 or appropriate dilution of the solvent (control) at 4 hpi and exposed 45 min to Tf-AF594 (µg/mL) followed by staining against either Rab5a or Rab11a and IE1 proteins. Representative images of intracellular distribution of internalized Tf-AF594 and Rab5a (A) or Rab11a (B) relative to the nuclear distribution of the IE1 protein. Inserts present zoomed area box. Bars, 10 µm. (C) 3D reconstruction of the Rab11a- and Tf-AF594-labeled TfRs in IN1-treated MCMV infected cells. The right panel presents the 3D view of the entire z-stack of 0.3 µm sections (32 slices) from the boxed area in Figure 4B using the Image J Volume Viewer plugin. The image shown on the right presents the 3D view of the entire stack, and the images on the left give the view across the vertical section through the stack. (D) Quantification of intracellular Tf-AF594 in control (Ø) and IN1-treated cells by ImageJ analysis. Dots represent the Total Corrected Cell Fluorescence (TCCF) of individual cells in the representative experiment (N = 3) and horizontal bars the median value. (E) Quantification of Rab5a and Rab11a intensity in control and IN1-treated cells by ImageJ analysis. Dots represent the Total Corrected Cell Fluorescence (TCCF) of individual cells in a representative experiment (N = 3) and horizontal bars the median value (F). The 3D colocalization of Tf with organelle markers and organelle markers with Tf in control and IN1-treated cells based on M1/M2 coefficients of pixel overlap measured across the Costes-algorithm thresholded z-stacks of confocal images. Data represent the mean ± SEM per cell (n = 8–12). Significance: * = p = 0.01, *** = p = 0.001.

Fig 4: Knockdown of GJA1 disrupted Rab11 trafficking to the ciliary base.(A) HA-tagged Rab11a was expressed in RPE1 cells, and the lysates were used for immunoprecipitation assays with GJA1 antibody-conjugated or IgG-conjugated beads. The immunoprecipitants were analyzed by immunoblotting with anti-GJA1 or anti-HA antibodies. Rab11a-HA was co-immunoprecipitated with endogenous GJA1. (B) RPE1 cells were serum-starved for 24 hr, and immunofluorescence staining analysis was performed with an anti-Rab11 antibody (green), an anti-GJA1 antibody (red), an anti-acetylated tubulin antibody (blue), and DAPI (gray). Rab11 and GJA1 accumulated around the pericentriolar-ciliary base. Scale bars: 5 µm. (C) GJA1-siRNA-treated RPE1 cells were immunostained with an anti-Rab11 antibody (green), an anti-GJA1 antibody (red), an anti-acetylated tubulin antibody (blue), and DAPI (gray). siRNA-mediated knockdown of GJA1 inhibited Rab11 accumulation around the pericentriolar region and basal bodies, and GJA1 transfection rescued the phenotype in RPE1 cells. Scale bars: 10 µm. (D) Statistical analysis of Rab11 accumulation in panel (C). Error bars represent the mean ± SD. P values were determined by one-way ANOVA (p***=0.0008, p*=0.0117). n=6(CTL, siRNA, Rescue), cell n=221(CTL), 157(siRNA), 133(Rescue). Raw values are provided in the Figure 7—source data 1 file. (E–F) Super-resolution images of Rab11 localization. RPE1 cells were labeled with an anti-Rab11 antibody (green) and an anti-acetylated tubulin antibody (red). GJA1 depletion was not observed in Rab11-positive ciliary vesicles in the pericentriolar region. Images were obtained by SIM. Scale bars: 5 µm (E), 1 µm (F). Figure 7—source data 1.The percentage of Rab11 accumulated cell. Figure 7—source data 2.The uncropped images of western blots.

Fig 5: PICALM interacts with Rab5 and Rab11 during Aß transcytosis across endothelial monolayera, Colocalization between PICALM (red) and Rab5 (green) in primary human brain endothelial cells (BEC) cultured with FAM–Aß40 (250 nM) for 2 min. b, Lack of association between PICALM (red) and Rab7 (green) in BEC cultured with FAM–Aß40 for 5 min. c, Colocalization between PICALM (red) and Rab11 (green) in BEC cultured with FAM–Aß40 for 5 min. Dapi, nuclear staining (blue). Insets: high magnification depicting colocalization. Bar=10 µm. d, Quantification of colocalization between PICALM and Rab5, Rab7, or Rab11 puncta in a–c. e–f, Colocalization of FAM–Aß40 (green) with Rab5 (magenta, upper) or RAB11b (magenta, bottom) 2 and 4 min after Aß internalization at the basolateral side of endothelial monolayer, respectively. Arrows denote co–localized white puncta. g–h, PLA of PICALM–Rab11 association (g, bar=10 µm) and kinetics of PICALM association with Rab5, Rab7 and Rab11 in endothelium after addition of Aß40 (1 nM) to the basolateral membrane (h). i, Coimmunoprecipitation of LRP1, Rab5, Rab7 and Rab11 by PICALM–specific antibody (IP: PICALM) 0, 2 and 4 min after addition of Aß40 (1 nM) to the basloateral membrane. j–k, Basolateral–to–apical transcytosis of Aß40 (1 nM) across monolayer expressing dominant negative Rab5–S34N, Rab7–T22N or Rab11b–S25N mutants compared to control EGFP (100%) over 60 min (j) and quantification of unidirectional Aß40 transport within 30 min (k). Mean ± s.e.m. from 3 primary isolates in triplicates. p<0.05 by ANOVA followed by Tukey’s posthoc test. l–m, Inhibition of Rab5 (l) and Rab11 (m) GTPase activity by si.PICALM compared to si.Scramble control.