Fig. 22

(Abstract P2). a Immunoprecipitation (IP), with anti-TβRII and control immunoglobulin G antibodies of EL4 cell lysates, followed by immunoblot of pulldown and input samples with the indicated antibodies. b Confocal microscopy images of EL4 cells stimulated with TGF-β (5 ng/ml for 1 h) and stained with anti-TβRII and anti-moesin antibodies. c and d HEK293FT cells co-transfected with plasmids encoding wild-type moesin-tagged with CFP at the carboxy terminus (Msn-WT-CFP) and TβRII-tagged with haemagglutinin at the carboxy terminus (TβRII-HA). Immunoprecipitation of solubilised proteins using anti-GFP and anti-HA antibodies and immunoblot of the pull-down samples. Input - whole cell lysate immunoblotting (throughout). e-g Immunoprecipitation and immunoblot (as in c) of HEK293FT cells co-transfected with CFP-tagged wild-type or phosphomimetic moesin mutants and TβRII-HA constructs. Data are representative of at least three (a-c) or four (d-f) independent experiments. h and i Primary CD4+ T cells (h) and B220+ B cells (i) from the spleen of WT and MsnKO mice were treated with cyclohexamide at the indicated times and surface TβRII analyzed by flow cytometry. Data represents the mean ± SD of at least three independent experiments. ***P <0.001 by two-way analysis of variance (ANOVA). j Flow cytometry analysis of primary CD4+ T cells isolated from the spleen of WT and MsnKO mice, and treated with brefeldin A (BFA), 20 μg/ml for up to 5 h and then washed. Cell surface TβRII was left to recover for up to 12 h prior to analysis. Data represents the mean ± SD of at least three independent experiments. ***P <0.001 by two-way analysis of variance (ANOVA)