Figure 1.

Cps1p is a stable integral membrane protein. (A) Cell lysates were fractionated on a 5% SDS-PAGE gel and subsequently subjected to immunoblotting with affinity purified anti–N- and C-terminal Cps1p antibodies (αCps1p) or with nonspecific antibodies (αGST) as a control. (B) Blots containing cell lysates were immunoblotted with antibodies specific to either N- or C-terminus of Cps1p. Antibodies were preincubated with antigens of N- or C-terminus peptides of Cps1p. (C) Cells were extracted after lysis in buffer containing 0.6 M NaCl, 0.1 M Na₂CO₃, 1.6 M urea, 4% Triton X-100, and 2% SDS. Soluble and insoluble proteins were separated by centrifugation as indicated by supernatant (s) and pellet (p), respectively. Proteins were fractionated using SDS-PAGE gels and immunoblotted using antibodies against Cps1p and Cdc8p. (D) Lysates were prepared from cells treated with 200 μM cycloheximide for the time intervals indicated and immunoblotted with antibodies against Cps1p.