FIGURE 8.

Subcellular localization of Kre6-3HA and Skn1-3HA. A, the lysate of cells carrying chromosomal KRE6-3HA at the original KRE6 locus (KTY284) was fractionated by sucrose density gradient centrifugation in the presence of 10 mm EDTA. Aliquots of fraction 1 (top) to 12 (bottom) were analyzed by SDS-PAGE, and proteins of interest were detected by immunoblotting using the antibodies, as shown on the right with filled triangles. Kre6-3HA was detected either by anti-Kre6 antibodies (Kre6) or by anti-HA monoclonal antibody (Kre6-3HA). Open arrowheads indicate bands of possible degradation products detected by anti-HA antibody. B, the lysate of cells carrying chromosomal SKN1-3HA at the original SKN1 locus (KTY432) was fractionated, and proteins of interest were detected by immunoblotting, as in A. C, the lysate of SKN1-3HA kre6Δ cells (KTY209) was fractionated and analyzed, as in B.