Journal: Nature immunology

Article Title: A p85α–osteopontin axis couples the ICOS receptor to sustained Bcl-6 expression by follicular helper and regulatory T cells

doi: 10.1038/ni.3050

Figure Lengend Snippet: p85α chaperones nuclear translocation of OPN-i. a, Immunoassay of lysates of 293T cells transfected with plasmids encoding Flag-p85α and OPN-i wild-type or OPN-i Y166F (YF) mutant, assessed by immunoprecipitation with anti-Flag and immunoblot analysis with anti-Flag and anti-OPN. Input, immunoblot analysis of an aliquot of cell lysates without IP. b, Confocal microscopy of OPN and Bcl-6 expression by CD44 + CD4 + T cells from OPN-i-KI mice 3 d post-injection with KLH in CFA, followed by cross-linking with anti-ICOS Ab (as in ) in vitro . Cells were counterstained with DNA-intercalating dye DAPI to trace nuclear perimeters. About 25–30 cells stained with Bcl-6 were further analyzed for localization of OPN protein. Right, fluorescence intensity expressed as the mean ratio of nuclear (Nuc) to cytoplasmic (Cyt) fluorescence pixel intensity ( n = 25–30 cells). c, Immunoblot analysis of nuclear fractions of OPN-i-KI CD62L − CD4 + T cells treated with anti-CD3 and/or anti-ICOS Abs (as in ), probed with anti-OPN, anti-Lamin B1 and anti-tubulin (to validate the integrity of nuclear separation). d , Immunoblot analysis of nuclear and cytosolic fractions of 293T cells transfected with plasmids encoding OPN-i wild-type or OPN-i Y166F mutant and increasing concentrations of Flag-p85α, probed with Abs, as indicated. e, Confocal microscopy of OPN and Bcl-6 expression by CD62L − CD4 + T cells from p85α WT or KO mice. Cell treatment and analysis as in a . Original magnification ( b, e ), 600×. * P < 0.05 and *** P < 0.001, Mann-Whitney test (error bars, mean ± s.e.m) (b, e ). All results are representative of at least two independent experiments.

Article Snippet: Antibodies or dyes used include: rabbit Bcl-6 (N-3), anti-rabbit Alexa Fluor 568 (for Bcl-6); mouse OPN (AKm2A1, Santa Cruz), anti-mouse Alexa Fluor 647 (for OPN) and nuclear dye DAPI.

Techniques: Translocation Assay, Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Confocal Microscopy, Expressing, Injection, In Vitro, Staining, Fluorescence, MANN-WHITNEY