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. 2014 Jul 14;33(16):1802–1814. doi: 10.15252/embj.201387076

Figure 7. PPS-negative auto-regulation.

Figure 7

  1. As in Fig6A, except that antibodies against the indicated proteins were used in the Western blot analysis.
  2. 20 μM PupE were incubated in pupylation buffer at 30°C together with 5 μM PafA (upper panel) or with 1 μM PafA and 5 μM 20S (lower panel). The first sample was removed (t = 0), ATP (2 mM) was added to start the reactions, and additional samples were removed at the indicated time points for SDS–PAGE followed by Coomassie Brilliant Blue staining.
  3. Reactions were mixed as in (B) with or without ATP or PupE, as indicated. Following a 2-h incubation at 30°C, aliquots were collected and subjected to SDS–PAGE followed by Coomassie Brilliant Blue staining.
  4. As in (A), except that a ΔprcSBA mutant was used that expresses from a chromosomally integrated plasmid either the prcS gene (designated -20S) or the prcSBA operon (designated +20S).
  5. Western blot analysis using antibodies against the indicated proteins was performed on samples collected at the indicated time points during a nitrogen starvation experiment that was performed as described in Fig6A.
  6. As in (D), except nitrogen source was re-supplemented following an hour of starvation.