FIGURE 6: The deletion of TOR1 increases Hog1p phosphorylation without affecting its cytosolic localization whereas SCH9 disruption diminishes Hog1p phosphorylation in isc1Δ cells.

(A) Hog1p activation was monitored in BY4741, isc1Δ, tor1Δ and isc1Δtor1Δ cells by immunoblotting, using anti-phospho-p38 antibody (top panel) that detects the phos-phorylated form of Hog1p, or anti-Pgk1p (loading control) as primary antibodies. A representative blot out of three is shown.

(B) S. cerevisiae BY4741, isc1Δ, tor1Δ and isc1Δtor1Δ cells expressing the consensus Rlm1p binding sequences fused to a LacZ reporter (2xRLM1-LacZ), were grown to the exponential phase and the β-galactosidase activity was measured as described in Materials and Methods. Values are mean ± SD of at least three independent experiments. ****p<0.0001; ***p<0.001; *p<0.05.

(C) Hog1p activation was monitored in BY4741, isc1Δ, sit4Δ and isc1Δsit4Δ cells by immunoblotting, as described in A. A representative blot out of three is shown.

(D) Hog1p activation was monitored in BY4741, isc1Δ, sch9Δ and isc1Δsch9Δ cells by immunoblotting, as described in A. BY4741 and sch9Δ cells were grown to the exponential phase and treated with either 10 μM C2-ceramide or DMSO (vehicle) for 1 h. A representative blot out of three is shown.