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. 1998 May 15;12(10):1464–1473. doi: 10.1101/gad.12.10.1464

Figure 2.

Figure 2

Figure 2

 (A) Localization of Wis1 MAPKK. Strain GD1892, bearing a genomic copy of wis1+ tagged with 12 copies of the myc epitope at the carboxyl terminus, was grown to mid-log phase at 30°C in YES medium. Cells were harvested before and after stress in YES + 0.6 m KCl at the indicated time and fixed in cold methanol. Wis1 was visualized by immunofluorescence using anti-myc antibody. Bar, 10 μm. (B) Strain FG2150 bearing a genomic copy of spc1+ tagged with two copies of the HA-epitope and six consecutive histidine residues on its carboxyl terminus, as well as a genomic copy of wis1+ tagged with 12 copies of the myc epitope, was grown to mid-log phase and harvested before and after KCl stress at the indicated times. Total cell homogenates were then prepared under native conditions, and Spc1 was purified using Ni2+–NTA–agarose beads. The precipitates were analyzed by immunoblotting after SDS-PAGE. Wis1 was detected with the anti-myc and Spc1 with anti-HA epitope antibodies.