FIGURE 1.

Cytoplasmic yeast two-hybrid analysis identifies AES as an interacting partner for NUP98-HOXA9. A, immunoblot showing expression of SOS-NUP98-HOXA9 in yeast cells. cdc25H yeast cells were transformed with either pSOS vector (Control) or pSOS-NUP98-HOXA9. Lysates were subjected to SDS-PAGE and immunoblotting with anti-HOXA9 antibody. Lysates from K562 cells expressing NUP98-HOXA9 were used as a positive control. B, K562 cDNA library in pMyr vector was co-transformed with pSOS-NUP98-HOXA9 into cdc25H yeast strain. Isolated colonies were patched on galactose-containing medium and transferred to 37 °C. Patches growing at 37 °C were re-spotted on plates containing selective medium supplemented with either glucose (repressing) or galactose (de-repressing) as the sole carbon source and incubated either at 23 (permissive) or 37 °C (restrictive) for 3–5 days. Plasmids were isolated from yeast cells growing at 37 °C in galactose containing medium and retransformed along with pSOS-NUP98-HOXA9 to confirm the interactions. Several positive cDNA clones were sequenced and identified as AES. Cells containing pSOS-NUP98-HOXA9 with pMYR-Lamin C or empty pSOS vector with pMyr-AES were spotted as negative controls. Cells containing pSOS-NUP98-HOXA9 with pMYR-SB were spotted as a positive control. Growth at 37 °C in galactose containing medium indicates a positive interaction.