Figure 2
Figure 2. Transcriptional and translational regulation of NKG2D and DAP10 by IL-2 and TGF-β1. Ex vivo isolated human NK cells were left untreated or were stimulated with IL-2, TGF-β1, or both IL-2 and TGF-β1 for either 1 or 3 days. (A) NKG2D (left panel) and DAP10 (right panel) transcripts were analyzed by qRT-PCR with specific primer sets and normalized to 18S rRNA. Scatter plots show mean values ± SEM. Each symbol represents a donor. Statistical significance was tested by paired Student t test and indicated by asterisks (*P < .05; **P < .01; ***P < .005). (B) Total cell lysates of primary NK cells were immunoblotted with anti-NKG2D (left panel) or anti-DAP10 (right panel) antibodies. (With commercially available antibodies, we are not able to detect NKG2D in the lysates of primary NK cells unless we concentrate the protein by some means, in this case TCA precipitation. The resultant high concentration of protein loaded on the gels leads to some distortion of the protein bands.) Actin was used as a loading control. The results are representative of 3 independent experiments with different donors. (C) The intensities of the upper band (left panel) and lower band (right panel) of DAP10 protein were quantified by densitometric analysis. Band intensities were normalized to actin and calibrated to values from untreated cells (value = 1). Data are shown as mean ± SEM from 3 different donors. Statistical significance is indicated by asterisks (*P < .05; ANOVA). (D) Ex vivo isolated human NK cells were stimulated for 3 days with either IL-2 alone or IL-2 with increasing amounts of TGF-β1. DAP10 transcripts were analyzed by qRT-PCR and normalized to 18S rRNA. Data from separate experiments with 7 different donors are presented as the percentage of DAP10 mRNA level in cells treated only with IL-2. Error bars represent SEM, statistical significance is indicated by asterisks (*P < .05; paired Student t test). (E) DAP10 protein levels in total cell lysates from ex vivo isolated human NK cells treated as in panel D were assessed by Western blot. A representative result of 3 different donors is shown. Actin was used as a loading control.

Transcriptional and translational regulation of NKG2D and DAP10 by IL-2 and TGF-β1. Ex vivo isolated human NK cells were left untreated or were stimulated with IL-2, TGF-β1, or both IL-2 and TGF-β1 for either 1 or 3 days. (A) NKG2D (left panel) and DAP10 (right panel) transcripts were analyzed by qRT-PCR with specific primer sets and normalized to 18S rRNA. Scatter plots show mean values ± SEM. Each symbol represents a donor. Statistical significance was tested by paired Student t test and indicated by asterisks (*P < .05; **P < .01; ***P < .005). (B) Total cell lysates of primary NK cells were immunoblotted with anti-NKG2D (left panel) or anti-DAP10 (right panel) antibodies. (With commercially available antibodies, we are not able to detect NKG2D in the lysates of primary NK cells unless we concentrate the protein by some means, in this case TCA precipitation. The resultant high concentration of protein loaded on the gels leads to some distortion of the protein bands.) Actin was used as a loading control. The results are representative of 3 independent experiments with different donors. (C) The intensities of the upper band (left panel) and lower band (right panel) of DAP10 protein were quantified by densitometric analysis. Band intensities were normalized to actin and calibrated to values from untreated cells (value = 1). Data are shown as mean ± SEM from 3 different donors. Statistical significance is indicated by asterisks (*P < .05; ANOVA). (D) Ex vivo isolated human NK cells were stimulated for 3 days with either IL-2 alone or IL-2 with increasing amounts of TGF-β1. DAP10 transcripts were analyzed by qRT-PCR and normalized to 18S rRNA. Data from separate experiments with 7 different donors are presented as the percentage of DAP10 mRNA level in cells treated only with IL-2. Error bars represent SEM, statistical significance is indicated by asterisks (*P < .05; paired Student t test). (E) DAP10 protein levels in total cell lysates from ex vivo isolated human NK cells treated as in panel D were assessed by Western blot. A representative result of 3 different donors is shown. Actin was used as a loading control.

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