Figure 3. Cdc34 S97 is phosphorylated in vivo and its detection is prohibited by certain protein extraction conditions.

A) Soluble protein from yeast cells, expressing from the CDC34 chromosomal locus, either wild type Cdc34 (lanes 1, 3 and 5), a COOH-terminally truncated Cdc34 encoding amino acids 1–244 (lane 2) or a Cdc34^tm mutant encoding the S97D mutation (lane 4) or bacterially expressed Cdc34^6XHis (lane 6) were analyzed by western blot using the α-pS97 phospho-specific or α-Cdc34 antibodies. B) An equal amount of soluble yeast protein extract made by either the glass bead or Horvath extraction protocol (see Materials and Methods) was analyzed by western blot using the α-pS97 phospho-specific and α-Cdc34 antibodies.