FIG. 4.

Pulse-chase labeling of de novo-synthesized SarA and SigB with [³⁵S]methionine upon ectopic overexpression of MazF_Sa. A culture of S. aureus ALC6094 harboring pG164-MazF(His₆) was induced at an OD₆₅₀ of 0.4 with IPTG (1 mM), while the noninduced culture received no IPTG. New protein synthesis was then monitored by isotopic labeling with [³⁵S]methionine for 15 min under induced or noninduced conditions at indicated intervals (0 to 90 min) after IPTG induction. The cells were then lysed and immunoprecipitated with anti-SigB monoclonal antibody (A) or anti-SarA monoclonal antibody (B) as described in Materials and Methods. Equivalent amounts of cell lysate, derived from equal culture volumes, were subjected to SDS-PAGE, followed by autoradiography. The labeled unknown protein that was immunoprecipitated by anti-SigB and anti-SarA antibodies served as the internal loading control.