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. Author manuscript; available in PMC: 2011 Jun 25.
Published in final edited form as: Immunity. 2010 Jun 25;32(6):743–753. doi: 10.1016/j.immuni.2010.06.002

Figure 7.

Figure 7

mTORC2 regulation of NF-κB activity via PKC. (A) Regulation of CD62L expression on T cells. Shown are FACS profiles of CD62L and CD44 expression on freshly isolated or activated (as in Fig. 4) CD4+ T cells (one experiment representative of four replicates). Inset numbers: frequencies (%) in each quadrant. (B) Rictor dependence of nuclear NF-κB subunits and the NF-κB-regulated protein Bcl-3 after TCR-CD28 costimulation. CD4+ T cells were activated and a portion of each was re-stimulated with αCD3 and αCD28 as in Fig. 6. After separation of nuclear (N) and cytosolic (C) fractions and resolution by SDS-PAGE, immunoblots were probed for the indicated species (one experiment representative of 2–3 replicates). Inset numbers: relative signals for each protein in the nuclei after normalization to lamin-A, with the nuclei of resting, previously activated WT cells set at 1. (C, D) PKC-θ reverses a defect mTORC2-deficient T cells in TCR-CD28 costimulatory induction of NF-κB transcriptional activity. (C) Activated CD4+ T cells were transfected with the RE/AP-luciferase reporter construct along with a constitutively active Renilla luciferase, and restimulated with αCD3 and αCD28. Relative activity: firefly luciferase measured 6 h after restimulation and normalized for transfection efficiency (one experiment representative of three replicates). (D) As in (C) except that cells also were co-transfected with empty vector or vector encoding the kinase-active PKC-θ mutant and the data are pooled from two independent replicates. (E) Decreased expression of Bcl-3. As in (A) except that the overall level of Bcl-3 was analyzed using whole-cell extracts. (F) mTORC2 promotes TCR-CD28-induction of ICAM-1 avidity. Using lymph node T cells ± stimulation with αCD3 and αCD28, activation- and avidity-dependent binding of Fc-ICAM-1-αFc complexes to CD4+ T cells was measured by flow cytometry. The frequencies (%) of ICAM-1-binding cells within the CD4+ gate are indicated by inset numbers, with results from two independent experiments summarized (right). Additional information is in supplemental Fig. S6.