PknB Phosphorylation Profile Figure 2. Verification of PknB-dependent phosphorylation of ATF-2. Recombinant ATF-2 was incubated with PknB (A) or p38 (B) in kinase reaction buffer for 30 minutes at 37uC. As a control, ATF-2 was incubated with PknB in the absence of ATP. After tryptic digestion, the resulting peptides were analyzed via online-mass spectrometry. The panels show the spectra for the ATF-2 peptide VIVADQTPTPTR that was either phosphorylated at the Thr73 by PknB, or Thr 69 and Thr 71 by p38. The b- and y-ions are high-lighted and the observed masses are given. Also the peptide sequence is indicated and amino acids that have been identified my mass spectrometric analysis are indicated in bold letters. The upper sequence corresponds to the b- and the lower sequence to the y-ions. doi:10.1371/journal.pone.0009057.g002 PLoS ONE | www.plosone.org 4 February 2010 | Volume 5 | Issue 2 | e9057