ppat.1006544.g007.tif (1.07 MB)

ZBTB32 and Blimp-1 bind cooperatively to target genes in activated CD8+ T cells.

Download (1.07 MB)
figure
posted on 21.08.2017 by Hyun Mu Shin, Varun N. Kapoor, Gwanghun Kim, Peng Li, Hang-Rae Kim, M. Suresh, Susan M. Kaech, E. John Wherry, Liisa K. Selin, Warren J. Leonard, Raymond M. Welsh, Leslie J. Berg

(a) WT CD8+ T cells pooled from three mice were stimulated with αCD3 and αCD28 for 2 days with or without a cocktail of cytokines containing IL-2, IL-4 and IL-12. Cells were harvested and stained with a mouse IgG and a rabbit IgG as negative controls (Iso), αBlimp-1 and αHDAC2, or αBlimp-1 and αZBTB32, followed by the Duolink proximity ligation assay. Samples were counter-stained for nuclei (blue; DAPI). Yellow signals demonstrate close proximity of the two proteins and data are representative of three independent experiments (left panels) and the graph (right panel) is a compilation of data from three independent experiments, and error bars represent the SEM. (b) WT CD8+ T cells pooled from three mice were stimulated with αCD3/CD28 for 2 days with or without a cocktail of cytokines containing IL-2, IL-4 and IL-12. Cells were harvested for immunoprecipitation with αZBTB32 or an IgG control antibody (left panel), followed by Immunoblotting (IB) with αBlimp-1. The presence of Blimp-1 and ZBTB32 in the whole cell lysates were confirmed (right panels). Data are representative of three independent experiments. WCL; whole cell lysate. (c) WT P14 cells were isolated from three recipient mice at day 7 post-LCMV-Armstrong infection, and chromatin was prepared. Primary ChIP assays were performed with antibodies to Blimp-1. Eluates from αBlimp-1 immunoprecipitates were re-precipitated with αZBTB32 or rabbit IgG (Iso) antibodies. Each ChIP eluate was amplified by Q-PCR for the indicated regions of the Eomes (left graph) and Cd27 (right graph) genes. (d-f) WT, Zbtb32-/- or Prdm1-/- P14 cells were isolated from three recipient mice at day 7 post-LCMV-Armstrong infection, and chromatin was prepared. ChIP assays were performed with antibodies to Blimp1, ZBTB32 or mouse IgG plus rabbit IgG (Iso). ChIP eluates were amplified by Q-PCR for the indicated regions of Eomes (d, upper), Cd27 (d, lower) and Il2ra (e). (f) Schematic of Il2ra gene loci. In each case amplicon 1 (Amp1) corresponds to putative Blimp-1 or ZBTB32 binding site and amplicon 2 (Amp2) to a negative control region. All graphs shown are from a compilation of three independent experiments and error bars represent the SEM.

History

Licence

Exports