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. 2016 May 25;7:11686. doi: 10.1038/ncomms11686

Figure 4. c-Cbl depletion augments but is not sufficient for NF-κB activation.

Figure 4

(a) Rested NKL cells transfected with control siRNA or c-Cbl-specific siRNA were stimulated through the indicated receptors for 5 min. Cell lysates were immunoblotted for phospho-p65 at serine 536 (pS536), phospho-p65 at serine 276 (pS276), p65, phospho-Akt at serine 473 (pS473), phospho-Erk1 and 2, c-Cbl or actin The normalized intensities of the phosphorylated p65 relative to p65 are presented. (b) Rested NKL-κB-GFP cells transfected with control siRNA or c-Cbl-specific siRNA were stimulated with plate-immobilized mAbs specific for NKG2D and/or 2B4 for 6 h. GFP expression in NKL-κB-GFP cells was analysed by flow cytometry, and representative result (top) and statistical bar charts (down) are shown. Values represent mean±s.d. (c) Cytokine release assays with rested NKL cells transfected with control siRNA or c-Cbl-specific siRNA and stimulated with NKG2D and/or 2B4 for 8 h. IFN-γ and MIP-1α in the supernatants were measured by ELISA. Values represent mean±s.d. *P<0.05; **P<0.01; ***P<0.001 (two-sided Student's t-test). Data are representative of at least three independent experiments.