Pmk1 activation in response to glucose deprivation is independent on the SAPK pathway and requires de novo protein synthesis. A. Strains MI200 (Pmk1-Ha6H; Control), and MI204 (sty1Δ, Pmk1-Ha6H), were grown in YES medium plus 7% glucose to early-log phase and transferred to the same medium with 3% glycerol. Aliquots were harvested at timed intervals and Pmk1 was purified by affinity chromatography. Either activated or total Pmk1 were detected by immunoblotting with anti-phospho-p44/42 or anti-HA antibodies, respectively. B. Control strain MI200 (Pmk1-Ha6H) was grown in YES medium plus 7% glucose to early-log phase, treated with of 100 μg/ml cycloheximide (CHX) for 60 min, and either transferred to the same medium with 3% glycerol (upper panel) or treated with 0.6 M KCl. Purification and detection of active or total Pmk1 was performed as described above.