Figure 2.
Expression of Mec1 derivatives with C-terminal mutations. (A) Amino acid sequences of the Mec1 derivatives. (B) Yeast strains CY6172 (Flag5-MEC1; lanes 1 and 2), CY6192 (Flag5-mec1-W2368A; lanes 3 and 4), CY6184 (Flag5-mec1-Δ1; lanes 5 and 6), CY6250 (Flag5-mec1-Δ2; lanes 7 and 8), CY6233 (Flag5-mec1-Δ3; lanes 9 and 10), CY6203 (Flag5-mec1-2369G; lanes 11 and 12), and BY4743 (lane 13) were grown in YPD media to mid-log phase and extracts were prepared by grinding in liquid nitrogen. Extracts were solubilized in the presence of protease inhibitors (1.0 mM phenylmethylsulfonyl fluoride, 5 μg/ml pepstatin, 1.0 mM benzamadine, 50 μg/ml trypsin inhibitor, and 5 μg/ml leupeptin); 50 μg and 10 μg protein were separated by sodium dodecyl sulfate (SDS)-PAGE; 50 μg was used for BY4743. The top portion of the gel was Western-blotted with anti-Flag (M2) antibody; the bottom portion was stained with Coomassie brilliant blue. (C) Interaction with Lcd1/Ddc2; 3 mg protein extract from yeast strains CY6172 (Flag5-MEC1; lanes 1 and 2), CY6192 (Flag5-mec1-W2368A; lanes 3 and 4), CY6184 (Flag5-mec1-Δ1; lanes 5 and 6), CY6203 (Flag5-mec1-2369G; lanes 7 and 8), and BY4743 (lane 9) containing YCplac111-myc9-Lcd1 were immunoprecipitated with anti-Flag antibody; 20 μl (odd-numbered lanes) and 5 μl of the immunoprecipitates were separated by SDS-PAGE and Western-blotted with anti-Flag antibody (upper panel) or anti-myc antibody (lower panel).