FIG. 3.
Phosphorylation of Slm1 and Slm2 is increased during heat stress when their calcineurin-dependent dephosphorylation is compromised. (A) GST-SLM2 (GBY115) and GST-SLM2ΔPEFYIE (GBY116) were expressed in a strain lacking slm1 and slm2. Cells were grown at 25°C in selective synthetic medium and expression was induced with 2% galactose. One culture was treated with FK506 (2 μg/ml) as indicated. Cells were shifted to 37°C and samples were taken at indicated time points (t). GST fusion proteins were purified from cell extracts and subjected to SDS-PAGE and Western blotting with anti-phospho and anti-GST antibodies. Immunoreactive bands were quantified using ImageJ software. The ratio of the anti-phospho signal over the anti-GST signal was plotted in arbitrary units (A.U.) as a function of time. (B) An approach similar to that described for panel A was taken for GST-SLM1 (GBY117) and GST-SLM1ΔPNIYIQ (GBY118).