Figure 3.
Optimizing the level of tetracycline during a chase. ubr1Δ S. cerevisiae carried the previously described (55), PRT-based plasmid pJO630 that expressed the Chk13f protein and the fDHFRha reference protein (see “Experimental procedures” and “Results and discussion” for a brief introduction of the mitotic checkpoint kinase Chk1, a physiological substrate of the Arg/N-end rule pathway). Cells were labeled for 3 min at 30 °C with 35S-EXPRESS Met/Cys at the specified times after the addition of Tc to the indicated varying final concentrations. Lanes 1–9, total 35S-labeling patterns, before immunoprecipitation. Lanes 10–18, same as lanes 1–9 but after immunoprecipitation of labeled cell extracts with anti-flag antibody (Ab). Lane 1, no addition of Tc before a 3-min pulse with 35S-EXPRESS Met/Cys. Lanes 2–6 and 10–15, Tc was added to cells to a final concentration of 0.1 mm, followed by incubation for 2, 4, 8, 15, and 20 min, respectively, before a 3-min pulse with 35S-EXPRESS Met/Cys. Lanes 7–9 and 16–18, Tc was added to cells to the final concentrations of 0.2, 0.3, and 0.5 mm, respectively, followed by incubation for 20 min and a 3-min pulse with 35S-EXPRESS Met/Cys. See “Results and discussion” for additional details.