Article Figures & Data
Figures
- FIGURE 1.
Distinct modes of GATA-3 gene expression exhibited by naive and differentiated T cells. A, For naive T cells (lanes 1–3), CD4+Mel-14high T cells were sorted from DO11.10 TCR-transgenic mice and activated as described (9 ) with the additions (+) of cytokines or Abs. Anti-IFN-γ (H22), 5 μg/ml, was included in all conditions. Anti-IL-12 (1:10) was added to all conditions except where IL-12 cytokine was added (lane 3). For effector analysis (lanes 4–8), T cells were primed either in Th0 conditions (anti-IL-12, 1/10 (23 ); anti-IL-4, 1/10 (22 ), and anti-IFN-γ (H22), 5 μg/ml (24 ), or in Th1 or Th2 conditions (9 ). For effector T cells, cells were harvested on day 7 and restimulated with plate-bound anti-CD3 Ab (TCR) (1/1000) in the presence (+) of the indicated cytokines or Abs. After 48 h, cells were harvested and analyzed by Northern blot. B, Th1 or Th2 effector cells as described in A were restimulated on day 7 after primary stimulation and restimulated with (+) or without (−) plate-bound anti-CD3 Ab (TCR), with (+) or without (−) addition of IL-4 or anti-IL-4 Ab.
- FIGURE 2.
Conservation of murine and human GATA-3 exon 1a and flanking sequences. The sequence of the exon 1a derived above (RACE) was aligned with homologous sequences identified from the murine and human genome databases from the Celera Discovery System. Sequence analysis for potential cis-regulatory elements was performed using Transfac resources (27 ).
- FIGURE 3.
Differential usage of GATA-3 exons 1a and 1b in mouse tissue and cell lines. A, Primers used for RT-PCT analysis. Primers a and c are specific for the 1a and 1b exons. Primer b is antisense to exon 2 and is used in both reactions. Primers 1a-AS and 1b-AS are antisense to exons 1a and 1b, respectively, and are used for hybridization to PCR products. B, RT-PCR analysis of exons 1a and 1b. RNA is from the indicated cells. Controls are shown either with no reverse transcriptase (lane 7), no RNA (lane 9) or positive control with plasmids (lane 8). C, RNA from T cells under Th2 differentiation was extracted at the indicated time course point, and RT-PCR was performed using the primers shown in A. D, RNA was harvested from naive cells (lanes 1 and 2) after 48 h stimulation with coated anti-CD3/CD28 in presence of Abs against IL-4 and IFN-γ (lane 1) or in presence of IL-4 with Ab against IFN-γ (lane 2). RNA from day 7 Th2 (lanes 3–5) or Th1 (lane 6) cells was harvested after 48 h of restimulation with splenic APCs and anti-IFN-γ, anti-IL-12 and IL-4 or anti-IL-4 Abs as indicated. As controls, RNA from thymus or brain in the presence or absence of reverse transcriptase (RT) reverse transcription as indicated (lanes 7–10), no RNA (lane 11), or plasmid-positive control (lane 12).
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