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. 2012 Jun 27;109(33):E2223–E2229. doi: 10.1073/pnas.1209115109

Fig. 5.

Fig. 5.

Activation of the Hippo pathway by CTLA-4 in vitro and in vivo. (A) Immunoblot analysis of lysates from activated OT-I cells cultured in the presence of antibodies to CD80 and CD86 and incubated for 1 h with beads bearing isotype control antibody or antibody to CTLA-4. Blots were developed with antibodies specific for phosphothreonine-183 of Mst1 and phosphoserine-909 of Lats1. Staining of actin is shown as a loading control. (B) Immunoblot analysis with antiphosphoserine-112 of Yap of lysates from activated OT-I cells expressing Yap S382A-HA that had been cultured in the presence of blocking antibodies to CD80 and CD86 and incubated for 1 h with beads bearing isotype control antibody or antibody to CTLA-4. HA staining is shown as a loading control. (C) Immunoblot analysis of WT Yap in cell lysates from OT-I cells activated in the presence of antibodies to CD80 and CD86 and incubated for 3 h with beads bearing various ratios of isotype control antibody and antibody to CTLA-4. Staining of actin is shown as a loading control. (D) Analysis by confocal microscopy of anti-Yap–stained OT-I cells that had been activated in the presence of blocking antibodies to CD80 and CD86 and incubated for 3 h with beads bearing isotype control antibody or antibody to CTLA-4 (green, anti-Yap; blue, DAPI). (Scale bars: 50 μm.) (E) Immunoblot analysis of Yap protein in lysates from CTLA-4−/− OT-I cells and WT OT-I cells that had been activated with SIINFEKL peptide and IL-2 for 24 h. (F) Analysis by confocal microscopy of Yap protein in CD8+ and CD4+ cells from the lymph nodes of CTLA-4−/− and CTLA-4−/+ 16- to 20-d-old mice. (Scale bars: 10 μm.)