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. 2004 Jan;15(1):71–80. doi: 10.1091/mbc.E03-08-0586

Figure 5.

Figure 5.

Slx4 associates with Slx1 to form an active endonuclease. (A) Slx1-TAP and Slx4-TAP fusion proteins were expressed in slx4-myc and slx1-myc strains, respectively. WCE, whole cell extract; IgG flow, flow through from WCE passed over IgG affinity column; TEV eluate, TEV cleavage products from IgG column; CaM flow, flow through from TEV eluate passed over calmodulin affinity column; CaM eluate, eluate from the calmodulin column. These fractions were subjected to immunoblotting revealed by 9E10 anti-myc monoclonal antibody and peroxidase-anti-peroxidase antibody (PAP; Sigma-Aldrich). Note: The loss of the TAP signal in the TEV-eluates and the following fractions is due to the loss of the protein A portion of the TAP-tag after TEV cleavage. (B) The SL substrate was incubated at 30°C for the indicated time with 3 μl of TEV-Slx1 or TEV-Slx4 obtained from slx1-TAP and slx4-TAP strains, respectively. Reaction products were analyzed by denaturing PAGE. (C) The SL substrate was incubated with 1, 2, 4, 6, and 9 μl of TEV-Slx1 obtained from slx1-TAP and slx1-TAP slx4 strains, for 30 min at 30°C, and reaction products were analyzed by denaturing PAGE.