FIG. 4.

Characterization of the mutant hshec1-113p. (A) Partial nucleotide and protein sequences of wild-type hsHEC1 and mutant hshec1-113 alleles. The nucleotide substitution in hshec1-113p (G to A) and the corresponding amino acid change at position 395 (Trp to a stop codon OPA) are underlined. (B) Identification of hshec1-113p in mutant cells. Cell lysate prepared from wild-type (lanes 1 and 2) and hshec1-113 (lanes 3 and 4) cells growing at 25°C (lanes 1 and 3) or 37°C (lanes 2 and 4) for 4 h were immunoprecipitated by mouse anti-hsHec1p MAbs, followed by SDS-PAGE and Western blotting with the same antibodies. (C) Subcellular localization of the mutant hshec-113p. Log-phase cells of hshec1-113 were grown at 25°C or shifted to 37°C for 2 h. hshec-113p was stained by purified rabbit anti-hsHec1p antibodies and Texas red-conjugated secondary antibodies. Nuclear DNA of the same cells was stained by DAPI; spindle was stained by rat antitubulin antibodies and FITC-conjugated secondary antibodies.