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. 2006 Mar;117(3):396–401. doi: 10.1111/j.1365-2567.2005.02313.x

Figure 1.

Figure 1

Yeast two-hybrid assay. The cytoplasmic domain of human CTLA-4 was used to screen a cDNA library generated from a HTLV-1-transformed T-cell line SLB-I, using a yeast two-hybrid system. Positive interaction was confirmed by expression of both the HIS3 and LacZ gene, thereby conferring ability on the positive clones to grow on media lacking His and to turn blue on media containing the chromogenic substrate X-Gal. Ten of the isolated clone inserts encoded 635 amino acids of STAT5a. To establish the specificity of this interaction, the plasmid encoding the 635 amino acid long segment of STAT5a was re-isolated from the library plasmid pACT2. This was then retransformed in yeast along with a vector encoding the cytoplasmic domain of CTLA-4 (CTLA-4 wt), CTLA-4 with a mutation at position 165 (CTLA-4 ms) or position 185 (CTLA-4 md) and CD28, respectively. Vectors encoding AP50 or Xenopus SMAD1 were used as positive and negative controls, respectively. Positive interaction was scored by the expression of HIS or LacZ. The relative β-galactosidase activity is indicated (blue colonies shown in grey). Two representative colonies are shown for each interaction.