Figure 7.
In vivo association of Ste50 and Ste11p is constitutive. Yeast strain YCW555 (MATa ssk2Δ ssk22Δ ste11Δ ste50Δ) was transformed with myc-tagged Ste11p expression construct (pCW199) and either a GST-Ste50p expression construct (pVL57) or a GST expression vector. Exponentially growing cells (3 × 109 cells) were induced with 0.5 mM CuSO4 (for the expression of GST-Ste50p or GST) for 2 h and then treated either with 3 μM α-factor for 1 h (A) or with 0.4 M NaCl for 10 min (B) before cells were harvested. Cell extracts were prepared and incubated with glutathione-Sepharose beads for 2 h at 4°C. The beads were washed, resuspended into sample buffer, and subjected to SDS-PAGE. Immunoblotting analyses were then performed with either anti-myc (9E10) monoclonal antibody or anti-GST polyclonal antibodies and detected with the ECL detection system.