Figure 7.

Detection by phosphate-affinity (Phos-tag) of the differentially phosphorylated forms of Slt2. (A) BY4741 slt2Δ-RLM1Myc cells containing the same plasmid pRS316 expressing Slt2 (WT), Slt2^T190A, Slt2^Y192F, or Slt2^{T190A Y192A} were grown to the mid-exponential phase in YPD and then stimulated with 30 µg/mL of CR for 2 h. Cell extracts were resolved by Phos-tag SDS–PAGE (top panel) and SDS–PAGE (bottom panel), and immunoblotted with anti-Mpk1 (Slt2) and anti-actin antibodies. (B) Cell extracts of slt2Δ-RLM1Myc cells containing plasmid pRS316 expressing the same proteins as in (A), as well as Slt2^T195V, Slt2^Y198F, Slt2^{T195V Y198F}, Slt2^CD3, and Slt2^KD, resolved as in (A) and immunoblotted with anti-Mpk1 (Slt2) and anti-actin (left panels), anti-Slt2-pY/pTpY (middle panel), and anti-Slt2-pT/pTpY (right panel) antibodies. A representative assay from three different experiments with distinct transformants is shown.