Figure 4.

Secondary methionine dependency test for the ChpAc.ΔN interacting candidates. Cdc25-2 cells were cotransfected with Met expression plasmid encoding for myc–ChpAc.ΔN protein bait and Yes expression plasmid encoding for oncogenic cytoplasmic Ras protein fused to rat pituitary cDNA library. 200 000 transformants were plated on 20 glucose plates lacking leucine and uracil and incubated at 24°C. Following the first replica plating ∼5% of the transformants exhibited efficient growth on galactose-containing medium at the restrictive temperature. Only 30 clones showed preferential growth on the galactose-containing plate lacking methionine. Subsequently, those transformants (only 20 are shown) were subjected to a secondary methionine test which resulted in the identification of two clones (indicated by *) that did not grow on the galactose plate containing methionine (right panel), but grew efficiently in the absence of methionine (left panel).