FIG. 2.

Identification of the RsbS and RsbRA isoforms separated by IEF. Wild-type and mutant cell extracts were analyzed by IEF, and the RsbS or RsbRA signals were detected with specific antibodies, as described in Materials and Methods. In all panels, gel images are oriented with their alkaline regions uppermost, toward the cathode, and the numbered lines to the right indicate the approximate positions of unmodified, singly modified, and doubly modified isoforms. (A) Lane 1, wild-type strain (PB2) (wt); lane 2, the rsbS deletion mutant (PB422) (ΔS). Wild-type extracts were also used for the λPP assays and were incubated for 18 h at 30°C (lanes 3 to 5). Lane 3, cell extract (CE) alone; lane 4, addition of λ reaction buffer and MnCl₂ (+λB); lane 5, further addition of λPP (+λPP). (B) Wild-type extracts are in lanes 1 and 4; mutant extracts are in lanes 2 and 3. Lane 2, strain with the RsbS S59A alteration (PB465); lane 3, RsbS S59D (PB477). (C) Lane 1, the wild-type strain (PB2) (wt); lane 2, the rsbRA deletion mutant (PB427) (ΔRA). Wild-type extracts were also used for the λPP assays shown in lanes 3 to 5, labeled as described for panel A. (D) Wild-type extracts are in lanes 1 and 8, and mutant extracts are in lanes 2 to 7. Lane 2, strain with the RsbRA T171A alteration (PB829); lane 3, RsbRA T171D (PB557); lane 4, RsbRA T205A (PB505); lane 5, RsbRA T205D (PB502); lane 6, RsbRA T171A-T205A (PB556); lane 7, RsbRA T171D-T205D (PB558).