Figure 6.

Hog1 interacts in vivo with various proteins of the replication complexes. Coimmunoprecipitation experiments were individually performed with the indicated TAP-tagged strains (Orc2, Mcm4, Cdc45, Psf2, Dpb2, or Cdc7). Each strain was transformed with either a control vector expressing GST or a vector expressing GST-Hog1. Cultures were treated with 200 mM HU for 2 h, osmostressed with 0.4 M NaCl for 10 min and then collected. One milligram of protein extracts was incubated with glutathione beads, extensively washed, eluted, and analyzed by SDS-PAGE. The resulting blots were probed with α-GST antibody to detect the immunoprecipitated GST or GST-Hog1 (α-GST, prec.) and α-TAP antibody to detect the coimmunoprecipitated proteins (α-TAP, prec.). Twenty micrograms of total protein extracts was run in parallel and probed with α-TAP (Total). Experiments were performed at least twice, and representative results are shown.