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. 2016 Nov 3;291(52):26707–26721. doi: 10.1074/jbc.M116.762179

FIGURE 3.

FIGURE 3.

Ets-2 and IL-2 expression in Jurkat cells. A, Jurkat cells were cultured in CM ± CsA ± P/I for a period of 4–48 h. Total RNA was isolated, cDNAs were prepared at the times indicated, and Ets-2 and IL-2 mRNA levels were measured by real time PCR using β2-m as an internal normalizer. B, Western blotting analysis of Ets-2 protein levels in Jurkat cells cultured in CM ± P/I ± CsA for a period of 4–16 h. Total protein cell extracts were prepared at the times indicated. Tubulin was used as an internal control for equal loading. Ets-2 protein levels were normalized according to tubulin levels and are also represented as -fold of expression with Ets-2 protein levels in CM ± CsA as baseline. C, Ets-2 silences IL-2 mRNA expression in Jurkat cells in a dose-dependent manner. Ets-2 and IL-2 mRNA expression was measured by real time PCR in Jurkat cells transfected with different amounts of a pcDNA3-ets-2 plasmid (0, 10, 20, and 30 μg) that overexpresses Ets-2. The amount of transfected DNA/sample was retained to 60 μg by the addition of appropriate amounts of vector pcDNA3 DNA. The cells were cultured in CM before transfection, retained for 6 h in CM after transfection to recover, and then transferred to CM + P/I for an additional 6 h. β2-m expression was used as an internal normalizer. D, Western blotting analysis to confirm expression of Ets-2 protein in transfected Jurkat cells. Jurkat cells were transfected with 20 μg of pcDNA3-ets-2 DNA for 6 h and then lysed for preparation of protein extracts. Tubulin was used as an internal control for equal loading. E, Jurkat cells were transfected with 2 μg of pLKO.1 vector or a mixture of five independent shRNA-ETS-2 clones and then cultured in CM ± P/I for 6 h. Total RNA was isolated, cDNAs were prepared, and IL-2 mRNA levels were detected by real time PCR using β2-m as an internal control. F, Western blotting analysis for verifying the silencing of Ets-2 performed with extracts prepared from transfected Jurkat cells with a mixture of the shRNA-ETS-2 clones cultured in CM ± P/I for 6 h. Tubulin was used as an internal control for equal loading. The results shown in A, C, and E are the mean values of three independent experiments; error bars represent S.D. (*, p < 0.05; **, p < 0.01; ***, p < 0.001, two-way ANOVA; *, p < 0.05, Student's t test). The results shown in B, D, and F are representative of three independent experiments.