One hundred μg of crude membrane protein were incubated with each substrate for 40 min on ice under UV irradiation (365nm). The samples were extracted and re-solubilized in RIPA/10% SDS before enrichment using neutravidin-agarose beads. Proteins were eluted, resolved by 10% SDS-PAGE, and visualized as described in Materials and Methods. A: His-Ste14p: lane 1: 200 μM 1, lane 2:100 μM 2, lane 3:100 μM 3, lane 4: 50 μM 7b, lane 5: 100 μM 7a , lane 6: 50 μM probe 8b, lane 7: 100 μM 8a, lane 8: empty vector (Δste14) + 100 μM 2. B:
His-hIcmt: lane 1: 200 μM 1, lane 2: 50 μM 2, lane 3: 50 μM 3, lane 4: 200 μM 7b, lane 5: 200 μM 7a, lane 6: 200 μM 8b, lane 7: 200 μM 8a, lane 8: empty vector (Δste14) + 50 μM 2.