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. Author manuscript; available in PMC: 2012 Mar 1.
Published in final edited form as: J Immunol. 2011 Jan 26;186(5):2959–2969. doi: 10.4049/jimmunol.1000112

FIGURE 4. Triad3A significantly reduces total 2DL4 levels and suppresses 2DL4-mediated cytokine production without impacting surface receptor expression in the KHYG-1 NK cell line.

FIGURE 4

A, Triad3A expression reduced the total level of 2DL4 protein in KHYG-1 cell lysates. KHYG-1 cells were transduced with FLAG-2DL4 ± FLAG-Triad3A and cell lysates were analyzed by immunoblotting with anti-Triad3A (top panel; top band is non-specific), anti-FLAG (middle) and anti-GAPDH (bottom). B, Surface expression of 2DL4 was not affected after co-transduction of Triad3A in KHYG-1 cells. KHYG-1 cells (assayed 2 days after IL-2 stimulation) that had been stably transduced with FLAG-2DL4 alone (shaded histograms) or in combination with FLAG-Triad3A (dashed line histograms) were stained for surface expression with anti-FLAG mAb plus PE-conjugated anti-κ secondary (antibody) or secondary alone (2ndary) by FACS. (C-E). KHYG-1 cells stably transduced with FLAG-2DL4 ± FLAG-Triad3A were harvested on day 2 after IL-2 stimulation, cultured for 24 hr (without IL-2) either with or without (NS) plate-bound anti-FLAG mAb (M2) or anti-NKp44 mAb (3.43.13), and analyzed by ELISA for secretion of IL-8 (C), IFN-γ (D), and MIP1-α (E). Results are representative mean ±S.D. from at least 3 experiments.