Fig 5. Pds1 and Smc1 are not required for the Esp1-Ty1-IN interaction.

(A) Quantitative PCR analysis of PDS1 expression driven from the TetO ₇ promoter after 100μg/mL doxycycline addition. The relative quantity of PDS1 transcript compared to a control (TAF10) is shown. (B) Esp1-GFP and untagged (No Tag) cells containing the TetO ₇-PDS1 allele and pGAL1-Ty1-H3 were induced for 24 hours with 2% galactose, then 100μg/mL doxycycline was added (+) or not (-) for one hour. Esp1-GFP was purified with GFP-Trap beads, immunoblot analysis performed and probed with anti-GFP and anti-IN (8b11) antibodies. (C) Esp1-GFP and untagged (No Tag) cells containing the TetO ₇—SMC1 allele and pGAL1-Ty1-H3 were induced for 24 hours with 2% galactose, then 100μg/mL doxycycline was added (+) or not (-) for six hours. Esp1-GFP was purified as in (B). The depletion of Smc1 was monitored with anti-Smc1 antibodies and the lysate was probed with anti-Pgk1 as a loading control.