Figure 6.

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.