Skip to main content
. 2009 May 19;284(28):18593–18604. doi: 10.1074/jbc.M109.018242

FIGURE 1.

FIGURE 1.

Identification of Mrc1 and Mec1, which cooperate in Rad53 activation. A, schematic of the RDS assay and its use to screen Rad53 activators. B, silver staining of inactive Rad53 and Dun1, purified from endogenously tagged Rad53-His6FLAG/rad9Δmrc1Δ cells and Dun1-TAF/rad53Δ cells, respectively. C, anti-FLAG immunoprecipitates from Mrc1-TAF/rad53Δ and rad53Δ cells stimulate the activation of Rad53. Anti-FLAG immunoprecipitates from mec1Δ, rad53Δ, and Mrc1-TAF/rad53Δ cells were added to the inactive RDS system to activate Rad53. The inactive RDS system consists of the following: 0.5 nm Rad53, 0.85 nm Dun1, and 3 μm GST-Sml1. Typically 1% of the anti-FLAG immunoprecipitate from two liters of yeast cell culture was used. Phosphorylated Sml1 was visualized using autoradiography and quantified using scintillation counting (see “Experimental Procedures” for details). These results are representative of three independent experiments. D, silver staining of 10% of the anti-FLAG immunoprecipitates from two liters of mec1Δ, rad53Δ, and Mrc1-TAF/rad53Δ cells. The band corresponding to Mec1 was identified by mass spectrometry.