Figure 1.
The α-P-Thr-169 antibody is specific for the Thr-169–phosphorylated form of Cdc28p. (A) Cdc28p-his6 purified from YKR101 was phosphorylated in vitro by GST-Cak1p (PO4-Cdc28p-his6) or treated with GST-Ptc2p and GST-Ptc3p (Cdc28p-his6). The indicated amounts of PO4-Cdc28p-his6, Cdc28p-his6, and unphosphorylated GST-Cdc28p purified from E. coli were immunoblotted with the α-P-Thr-169 antibody. The membrane was then stripped and reprobed with an α-PSTAIR antibody to detect total Cdc28p. The asterisk (*) denotes degradation products of GST-Cdc28p. (B) Extracts (20 μg of protein) from strains YKR102 (Cdc28p-HA; WT) and YKR103 (Cdc28pT169A-HA; T169A) were immunoblotted with the α-PSTAIR, α-HA, and α-P-Thr-169 antibodies. Note that both strains contain endogenous Cdc28p as well as the indicated form of HA-tagged Cdc28p. (C) Extracts from strains YKR104 (Δcak1) and YKR105 (CAK1) were fractionated on a Superdex 200 column. Fractions 16 and 17, which contained the monomeric Cdc28p, were immunoblotted with the α-PSTAIR and α-P-Thr-169 antibodies. Cdc28p43244-HA is a mutant form of Cdc28p that can function in the absence of activating phosphorylation (Cross and Levine, 1998).