FIGURE 3:

Ded1 phosphorylation and/or misregulation of downstream TORC1 signaling are not the cause of ded1-∆CT rapamycin resistance. (A) Sch9::3xHA cells harboring DED1 or ded1-∆CT were subjected to a time course of rapamycin treatment, NTCB chemical cleavage of cell extracts, and blotted for with 12CA5 antibody (∝HA) to visualize the band-shift. A representative blot is shown along with a graph of the means from three independent trials. (B) DED1 or ded1-∆CT cells were treated for 1 h with 200 ng/ml rapamycin, and cell extracts were prepared and blotted with antibodies against eIF2α, p-eIF2α, and PGK1. (C) Cells containing DED1, ded1-4SA (S535/S539/541/543A: phospho-deficient), or ded1-4SD (S535/539/541/543D: phospho-mimetic) were serially diluted onto rich media ± rapamycin (200 ng/ml) and incubated at 30°C. (D) DED1 or ded1-∆CT, plasmids were shuffled into WT or tif4631∆ (eIF4G1-null) strains. Cells were serially diluted onto YPD ± rapamycin (200 ng/ml) and incubated at 30°C.