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. 2007 Nov;18(11):4405–4419. doi: 10.1091/mbc.E07-05-0484

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© 2007 by The American Society for Cell Biology

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Figure 5. — Sty1p hyperactivation promotes defective Pmk1p activation under saline stress. (A) Sty1p basal activity in different mutants. Strains JM1521 (sty1-HA6H, Control), MI1001 (sty1-HA6H, pyp1Δ), MI1002 (sty1-HA6H, pyp2Δ), MI1003 (sty1-HA6H, ptc1Δ), MI1000 (sty1-HA6H, pmp1Δ), and MI100 (sty1-HA6H, pmk1Δ) were grown in YES medium to mid-log phase, and Sty1-HA6H was purified by affinity chromatography. Activated or total Sty1p was detected by immunoblotting with anti-phospho-p38 and anti-HA antibodies, respectively. (B) Increased Pyp2p and Ptc1p synthesis in pyp1Δ cells. Strains MI702 (pyp2-13myc, Control) and MI704 (pyp2-13myc, pyp1Δ) (top), MI703 (ptc1-13myc, Control), and MI705 (ptc1-13myc, pyp1Δ) (bottom) were grown in YES medium to mid-log phase and treated with 0.6 M KCl. Aliquots were harvested at the times indicated, and total extracts were obtained. Pyp2-13myc and Ptc1p-myc fusions were detected by immunoblotting with anti-c-myc antibody. Anti-Cdc2 antibody was used as loading control. (C) Pmk1p activation in wis1DD cells. Strains MI200 (pmk1-HA6H, Control), MI709 (pmk1-HA6H, wis1DD), and MI713 (pmk1-HA6H, wis1DD, atf1Δ) were grown in YES medium to mid-log phase and treated with 0.6 M KCl. Aliquots were harvested at the times indicated and Pmk1-HA6H was purified by affinity chromatography. Activated Pmk1p was detected by immunoblotting with anti-phospho-p42/44 and total Pmk1p with anti-HA antibodies. (D) Increased Pyp1p, Pyp2p and Ptc1p synthesis in wis1DD cells. Strains MI701 (pyp1-13myc, Control) and MI710 (pyp1-13myc, wis1DD) (top), MI702 (pyp2-13myc, Control) and MI711 (pyp2-13myc, wis1DD) (middle), MI703 (ptc1-13myc, Control) and MI712 (ptc1-13myc, wis1DD) (bottom) were grown in YES medium to mid-log phase and treated with 0.6 M KCl. Aliquots were harvested at the times indicated, and total extracts were prepared. Pyp1-myc, Pyp2-13myc, and Ptc1p-myc fusions were detected by immunoblotting with anti-c-myc antibody. Anti-Cdc2 antibody was used as loading control.