FIG. 4.

Coimmunoprecipitation of Swi4 with mutant forms of Mpk1 in response to heat shock and caffeine treatment. Yeast strain DL456 (mpk1Δ) was cotransformed with plasmids bearing the indicated FLAG-tagged MPK1 allele (wild-type MPK1 [p2704], mpk1-R196A [p2706], and mpk1-K83A [p2709]) and HIS-tagged SWI4 (p2418). Transformants were cultivated to mid-log phase in selective medium lacking methionine (to induce expression of Swi4-6HIS) and either maintained at 25°C or subjected to cell wall stress by changing the temperature to 39°C (+) or by adding 8 mM caffeine for 2 h (+). Cell extracts (lysate) and immunoprecipitates (IP) with anti-FLAG M2 affinity gel were subjected to SDS-PAGE and analyzed by immunoblotting with anti-FLAG antibodies to detect Mpk1 or with anti-HIS antibodies to detect Swi4.