FIG. 5.

Tagging of Skp1p at the endogenous locus. (A) Cells transformed to integrate a C-terminal TAP or ZZ tag at SKP1 were selected to obtain maximal replacement of the wild-type (WT) locus and then released from selective pressure. Genomic DNA was isolated from wild-type cells and cells from independent clonal isolates of each gene replacement, digested with ClaI and HpaI, and used for Southern blot hybridization with a DNA probe equally complementary to wild-type and tagged SKP1 loci. A separate lane of the same nondenaturing gel was loaded with a DNA ladder. (B) Whole-cell extracts from the same cells were normalized by total protein concentration prior to protein analysis by immunoblotting. Markers were run in the same gel and transferred to the same blot. (C) Genomic DNA from the same cells was digested with HindIII and analyzed by Southern blotting for the terminal restriction fragment of macronuclear rDNA chromosomes. One lane of the denaturing polyacrylamide gel was loaded with an end-labeled DNA ladder.