Fig 2: Rab11−FIP1 colocalizes with pIgA during pIgA transcytosis from the vicinity of centrosome to apical plasma membrane. (A) Colocalization of Rab11−FIP1 with the centrosome was detected. Vero−pIgR cells (1 × 105) were grown on Transwell for 3 days. Cells were fixed with 4% paraformaldehyde and stained with the indicated antibodies before observation by confocal microscopy. Scale bar: 10 µm. (B,C) Colocalization of Rab11−FIP1, pIgA with the Golgi apparatus (GM130) or recycling endosomes (Rab11a) was detected. Vero−pIgR cells (1 × 105) were grown on Transwell for 3 days. An amount of 20 µg pIgA was added or not added to the basal chamber for 10 min at 37 °C and cells were then washed three times. Subsequently, cells were cultivated at 37 °C and harvested at the indicated time points. Finally, cells were fixed with 4% paraformaldehyde and stained with the indicated antibodies before observation by confocal microscopy. Scale bar: 10 µm. (D,E) Quantitative analysis of colocalization of Rab11−FIP1, pIgA with GM130 or Rab11a. Statistical analysis was based on colocalization images (covering dozens of cells) using the ImageJ software. (F) Analysis of localization of Rab11−FIP1 on the endosomes containing pIgA. Vero−pIgR cells (1 × 105) were grown on Transwell for 3 days. An amount of 20 µg pIgA was added to the basal chamber for 15 min at 37 °C and cells were then washed three times. Cells were fixed with 4% paraformaldehyde and then were permeabilized by 0.1% Triton X−100 or 20 µg/mL digitonin for 10 min at 4 °C. Finally, cells were stained with the indicated antibodies before observation by confocal microscopy. Scale bar: 10 µm. Data of (A–F) are representative of three independent experiments.

Fig 3: TRIM21-mediated polyubiquitination of Rab11-FIP1 and Rab11-FIP5 is crucial for their ability to facilitate pIgA transcytosis. (A) Effects of pIgA transcytosis on the ubiquitination of Rab11-FIP1. Vero-pIgR cells (4 × 105) were grown on 24 mm diameter Transwell (0.4 µm pore) for 3 days. An amount of 80 µg pIgA was added or not added to the basal chamber for 1 h. The cells (2 × 107) were harvested for ubiquitination assays with the indicated antibodies. (B) The interaction between Rab11-FIP1, Rab11-FIP5 and TRIM21 was detected. HEK293T cells (2 × 106) were co-transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. (C) TRIM21 mediated polyubiquitination of Rab11-FIP1 and Rab11-FIP5 was detected. HEK293T cells (2 × 106) were co-transfected with the indicated plasmids for 24 h. Ubiquitination assays were performed with the indicated antibodies. (D) Effects of TRIM21 knockdown on the K11-linked ubiquitination of Rab11-FIP1 during pIgA transcytosis. Vero-pIgR cells were transduced with control or the indicated TRIM21-RNAi plasmids by retroviral-mediated gene transfer to establish stable cell lines. The indicated Vero-pIgR cells (4 × 105) were grown on 24 mm diameter Transwell for 3 days. An amount of 80 µg pIgA was added or not added to the basal chamber for 1 h. The cells (2 × 107) were harvested for ubiquitination assays with the indicated antibodies. (E) Effects of TRIM21 knockdown on pIgA transcytosis. The control and TRIM21-RNAi Vero-pIgR cells (1 × 105) were grown on Transwell for 3 days. An amount of 20 µg pIgA was added or not added to the basal chamber for 10 min at 37 °C and cells were then washed three times. Subsequently, cells were cultivated at 37 °C and harvested at the indicated time points. Finally, cells were fixed with 4% paraformaldehyde and stained with the indicated antibodies before observation by confocal microscopy. Scale bar: 10 µm. (F) Effects of reconstitution of TRIM21-knockdown cells with TRIM21 but not its LD mutant on extracellular secretion of pIgA. The control and TRIM21-RNAi Vero-pIgR cells transduced with the indicated plasmids were grown on Transwell for 3 days. An amount of 20 µg pIgA was added to the basal chamber for 4 h. The supernatant was collected for IgA analysis by ELISA. (G) The knockdown and reconstitution efficiencies of TRIM21 in the cells were detected by immunoblotting analysis. (H) Effects of TRIM21 knockdown on extracellular secretion of pIgA in polarized Vero-pIgR cells. The indicated Vero-pIgR cells (1 × 105) were grown on Transwell for 6 days. An amount of 20 µg pIgA was added to the basal chamber for 2 h. The supernatant was collected for IgA analysis by ELISA. Data of (F) were analyzed by Kruskal-Wallis one-way ANOVA. Data of (H) were analyzed by unpaired, two-tailed Student’s t-test. Graphs show mean ± SD; n = 3. ** p < 0.01. Data of (A–H) are representative of three independent experiments. EV: empty vector. WT: wild type plasmid. LD: ligase-dead (LD) mutant plasmid.

Fig 4: Rab11-FIP1 interacts with pIgR. (A) HEK293T cells (2 × 106) were transfected with pIgR-BirA* or BirA* plasmids for 24 h. Cells were untreated or treated with 50 µM biotin for 12 h before lysis in the indicated buffer. Biotin-labelled proteins were immunoprecipitated from lysates using streptavidin conjugated agarose beads. Immunoprecipitated proteins were detected by immunoblotting analysis and identified by mass spectrometry. (B) The samples of (A) were respectively detected by immunoblotting analysis with the indicated antibodies. (C) Detection of the interaction between Rab11-FIP1 and pIgR. HEK293T cells (2 × 106) were co-transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. (D) Domain mapping of the interaction between Rab11-FIP1 and pIgR. HEK293T cells (2 × 106) were co-transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. ECD: ectodomain; TM: transmembrane domain; CPD: cytoplasmic domain. ++: stronger interaction, +: interaction, -: no interaction. Data of (A–D) are representative of three independent experiments.

Fig 5: The Rab11-FIP1, Rab11-FIP5 and pIgR complex facilitates pIgA transcytosis. (A) The interaction between Rab11-FIP5 and pIgR was detected. HEK293T cells (2 × 106) were co-transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblot analyses were performed with the indicated antibodies. (B) Domain mapping of the interaction between Rab11-FIP5 and pIgR. HEK293T cells (2 × 106) were co-transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. (C) The interaction between Rab11-FIP1 and Rab11-FIP5 was detected. HEK293T cells (2 × 106) were co-transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. (D) Colocalization of Rab11-FIP1, Rab11-FIP5 and pIgR was detected. Vero-pIgR cells (1 × 105) were co-transfected with GFP-Rab11-FIP1 (0.2 µg) and Cherry-Rab11-FIP5 (0.2 µg) for 24 h. The transfected cells were fixed with 4% paraformaldehyde and stained with the indicated antibodies before observation by confocal microscopy. Scale bar: 10 µm. (E) Colocalization of Rab11-FIP1, Rab11-FIP5 and pIgA was detected during pIgA transcytosis. Vero-pIgR cells (1 × 105) were grown on Transwell for 3 days. An amount of 20 µg pIgA was added or not added to the basal chamber for 10 min at 37 °C and cells were then washed for three times. Subsequently, cells were cultivated at 37 °C and harvested at the indicated time points. Finally, the cells were fixed with 4% paraformaldehyde and stained with the indicated antibodies before observation by confocal microscopy. Scale bar: 10 µm. Data of (A–E) are representative of three independent experiments.