[PMC free article] [PubMed] [Google Scholar] 38. BCR signaling is also required for BCR ubiquitination and BCR-mediated antigen processing and demonstration. Knockdown studies expose that AICAR phosphate of the two known Syk-binding E3 ubiquitin ligases c-Cbl and Cbl-b, only c-Cbl appears to have a central part in BCR ubiquitination, trafficking to MIIC, and ubiquitin-dependent BCR-mediated antigen processing and demonstration. These results set up the novel part for Syk signaling and the Syk-binding ubiquitin ligase c-Cbl in the BCR-mediated processing and demonstration of cognate antigen and define one mechanism by which antigen-induced BCR ubiquitination is definitely modulated to effect the initiation and maturation of the humoral immune response. indicate the shRNA sense binding sites. test was used to compare the level of Cbl in knockdown WT cells. *, 0.05. Anti-c-Cbl and Cbl-b Western Blotting B cells Rabbit Polyclonal to POLE4 (WT, c-Cbl, Cbl-b, GAPDH, and Scrambled) were lysed at 1 107 viable cells/ml in radioimmunoprecipitation assay buffer for 10 min on AICAR phosphate snow. Postnuclear supernatants were generated by centrifugation for 15 min at 16,000 at 4 C. Samples were analyzed by SDS-PAGE and Western blot analysis (8% gel) using damp transfer conditions. Antibodies anti-c-Cbl (catalog 2747, Cell Signaling Technology) and anti-Cbl-b (clone C20, catalog sc-1435, Santa Cruz Biotechnology) were used to probe for these 120-kDa proteins. -Actin was used as a loading control. Immunofluorescence Microscopy A20WT B cells and knockdown cells were pulsed AICAR phosphate with 10 g/ml biotinylated Rb-anti-huIgM-btn on snow and washed twice to remove excessive ligand. Cells were then stained with SA-Alexa Fluor 594. AgBCR complexes were then allowed to internalize at 37 C for 0C60 min. Cells were attached to Alcian blue-treated coverslips, fixed, and permeabilized (or not) as explained previously AICAR phosphate (29). For staining of the Light-2 positive compartment, cells were permeabilized in 0.01% saponin and stained with anti-LAMP-2 (1:100) and anti-rat IgG (H+L) Alexa Fluor 647. Nuclei were stained with 1 g/ml DAPI before final washing and mounting with Fluoro-gel (Electron Microscopy Sciences) mounting press. Samples were visualized with an Olympus Fluoview FV1000 microscope (60 numerical aperture 1.25 water immersion lens). Measuring Colocalization Quantiation of BCR/Light-2 colocalization was carried out by determining the Pearson’s coefficient, which actions the strength of the linear relationship between two variables, for each sample. The analysis was applied to all Z-stack slices. For each condition, 100C150 cells were analyzed. In Vitro Antigen Control and Demonstration Assay B cells (WT or knockdowns) and Ova-specific T cells (DO11.10) were cocultured at a 1:1 percentage with an indicated concentration of either Ova or PC-Ova for 24 h at 37 C inside a 96-well plate. After 24 h, supernatants were collected, and IL-2 levels were identified using a mouse IL-2 ELISA (Ready-SET-Go! eBioscience, catalog no. 88-7024) Statistical Analysis Data were analyzed using Student’s test for variations in densitometry (Figs. 3 and ?and5),5), BCR endocytosis (Fig. 4), and antigen demonstration (Figs. 2 and ?and6)6) using Microsoft Excel 2008 for Mac pc Version 12.3.0. Statistical significance is definitely indicated as 0.05. Open in a separate window Number 2. BCR-mediated antigen demonstration requires Src-family kinase and Syk signaling. test. *, 0.05. and = 0.75). Open in a separate window Number 5. c-Cbl mediates AgBCR ubiquitination and processing. and = 10 m. test was used to compare the Pearson’s coefficient of knockdown WT cells. *, 0.05. RESULTS Syk Is Required for Antigen-induced BCR Ubiquitination and BCR-mediated Antigen Control BCR connection with cognate antigen results in lipid raft-dependent BCR signaling including raft-resident Src-family kinases and the downstream kinase Syk. BCR ubiquitination, which is critical for the trafficking of AgBCR complexes to MVB-like antigen processing compartments, happens via an Src-family kinase-dependent mechanism and is restricted to lipid raft-resident AgBCR complexes (18, 31). Although Syk activity has been implicated in BCR-mediated antigen processing and demonstration (5), the part of Syk in BCR ubiquitination remains unclear. To establish the part of Syk in BCR ubiquitination, B cells were treated with the Syk inhibitor piceatannol, AICAR phosphate and the impact on AgBCR ubiquitination was identified. As expected, treatment of either the A20WT B cell collection (Fig. 1and from the whole cell lysate and ubiquitinated BCR and from your UQ1 pull-down) was recognized by SDS-PAGE and anti-IgM Western blot analysis. Ligand-BCR (from the whole cell lysate and ubiquitinated AgBCR and from your UQ1 pull-down) was recognized by SDS-PAGE and.
[PMC free article] [PubMed] [Google Scholar] 38