Figure 4.

Clb5 and Clb6 protein accumulation is delayed in response to osmotic shock. (a and b) Clb5 and Clb6 protein expression are delayed in osmostressed cells. Exponentially growing sic1 cells with endogenously TAP-tagged Clb5 (a) or Clb6 (b) were synchronized in α-factor for 3 h and released into fresh YPD medium. Cells were either not osmostressed (control) or stressed with 0.4 M NaCl 30 min (t₃₀) after α-factor release as indicated by the arrowhead. Samples were analyzed by SDS-PAGE and probed with α-TAP antibodies to detect endogenous Clb5-TAP and Clb6-TAP levels (top part of each panel). Blots were then stripped and probed with α-PSTAIR to detect Cdc28 protein levels (bottom of each panel). (c) Clb5 protein accumulation during S phase is delayed in a Hog1-dependent manner. TAP-Clb5 tagged sln1^ts4 sic1Δ with the wild-type HOG1 allele (bottom) or a hog1Δ deletion (top) were grown, synchronized with α-fac, and released into fresh medium. The cultures were shifted to 37°C after their release from α-fac. Samples were analyzed for Clb5 and Cdc28 levels as described in a and b. Experiments were performed at least twice, and representative results are shown. (d) Hog1 activation does not delay the Dbf4 protein accumulation during S-phase. sln1^ts4 sic1 cells containing the wild type HOG1 allele (top) or hog1 deletion (bottom) were grown, synchronized with α-fac, and released into fresh medium and shifted to 37°C. Samples were analyzed by SDS-PAGE and blotted and probed with specific polyclonal antibodies to detect the endogenous levels of the Dbf4.