FIGURE 1.

Immunoblot of His-Ste14p cross-linked chemically with BS³. A, 80 μg of crude membrane protein were incubated with 0.8 mm BS³ for 20 min at room temperature. The reaction was terminated by the addition of SDS-PAGE sample buffer without reducing agents and then heated for 20 min at 65 °C. Various amounts of crude membrane proteins were separated by 10% SDS-PAGE, and His-Ste14p was detected using a Ste14p polyclonal antibody (1:1,000) and a HRP-conjugated secondary goat anti-rabbit antibody (1:10,000) as follows: lane 1, 1 μg of His-Ste14p (CH2704); lane 2, 1 μg of His-Ste14p (CH2704) + BS³; lane 3, 10 μg of untagged Ste14p (CH2866; 2μ PGK promoter); lane 4, 10 μg of untagged Ste14p (CH2866; 2μ PGK promoter) + BS³; lane 5, 50 μg of SM3495 (untagged Ste14p; 2μ STE14 promoter); lane 6, 50 μg of SM3495 (untagged Ste14p; 2μ STE14 promoter) + BS³; lane 7, 50 μg of CH2714 (negative control); lane 8, 50 μg of CH2714 (negative control) + BS³. Protein bands were visualized using enhanced chemiluminescence (ECL). B, purified His-Ste14p (2.5 μg) was incubated with 0.4 mm BS³ for 20 min, and the reaction was terminated by the addition of SDS-PAGE sample buffer without reducing agents. The reactions were heated for 30 min at 65 °C, 0.1 μg of purified protein was separated by 7.5% SDS-PAGE, and the protein bands were visualized by immunoblot analysis as described above.