FIG. 1.

Western blot analysis of wild-type and mot1-1 nuclear extracts. (A) Specificity of antiserum and comparison of native and recombinant MOT1. The two panels were loaded with identical protein samples; the left-hand panel was probed with preimmune serum, and the right-hand panel was probed with anti-MOT1 antiserum. Lanes 1 and 2 contain 10 μg of nuclear extract from wild-type and mot1-1 yeast cells, respectively. Lane 4 contains 250 ng of rMOT1; lane 3 contains a comparable amount of yMOT1 whose activity was not determined. The arrow indicates the position of full-length immunoreactive MOT1. (B) Quantitation of MOT1 in wild-type nuclear extract. Lane 1 contains 10 μg of total nuclear extract protein. Lanes 2 to 6 contain 90, 120, 210, 240, or 300 ng, respectively, of recombinant MOT1. The blot was probed as described for panel A, with anti-MOT1 antiserum. (C) Quantitation of MOT1 and TBP in wild-type or mot1-1 nuclear extract. Lane 1 contains 20 μg of wild-type nuclear extract protein. Lanes 2 and 3 contain 20 μg of total protein from two independently prepared samples of mot1-1 nuclear extract. Lanes 4 to 11 contain MOT1 purified from a yeast overexpression strain (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, or 5.0 U, respectively). Lanes 12 to 16 contain 1, 3, 9, 15, or 20 ng, respectively, of recombinant yeast TBP. The blot was probed with anti-MOT1 (top half) or anti-TBP (bottom half) rabbit polyclonal antiserum.