DRP-1b - A Novel ZIPk-Like Isoform Figure 7. The DAP kinases phylogenetic tree. A phylogenetic tree of the indicated organisms was constructed based on the multiple alignment of the DAP kinase scatalytic domain, using the PHYML program. Numbers above branches represent bootstrap support from 100 replicates. Yellow background- high bootstraps value. Blue- DAPk; Green- DRP-1; Red- ZIPk; Black- ortholog undetermined due to partial sequence. doi:10.1371/journal.pone.0017344.g007 blot analysis was conducted to ensure equal kinase protein expression. Immunoprecipitation Cells were washed twice in PBS and then suspended and vortexed in cold PLB lysis buffer (5 mM EDTA, 10 mM NaPO4, 1% Triton X-100, 0.1 M NaCl, 0.5% DOC, 0.1% SDS) with protease inhibitors (1% protease inhibitor cocktail (Sigma), 1% PMSF). Lysates were centrifuged for 15 min. at 14,000 rpm at 4uC. The pellet was discarded and the supernatant was pre-cleared for 1 hour at 4uC on a slurry of protein G-PLUS Agarose beads (Santa Cruz Biotechnology). The pre-cleared extracts were incubated with Agarose-conjugated anti-FLAG M2 gel beads (Sigma) for 2 hours at 4uC. Immunoprecipitates were washed 4 times with lysis buffer containing protease inhibitors, and resolved by standard SDS-PAGE. Blots were reacted with anti-FLAG-M2 monoclonal antibody (dilution 1:500) (Sigma); anti-CREB-2 (ATF4) rabbit polyclonal antibody (dilution 1:500) (Santa Cruz); or anti-Actin monoclonal antibody (dilution 1:5000) (Sigma). In vitro kinase assay Immunoprecipitated FLAG- DRP-1b, DRP-1 or ZIPk were quantified after elution against standards. 200 nmol bacterial expressed, purified, human MLC was incubated with or without 50 nmol kinase in reaction buffer (50 mM HEPES, pH 7.5, 20 mM MgCl2) containing 1 mM bovine calmodulin, 0.5 mM CaCl2 and 50 mM ATP. The kinase assay was conducted at 30uC for 30 minutes. Protein sample buffer was added to terminate the reaction, and after boiling, the proteins were analyzed resolved by standard SDS-PAGE. Kinase protein levels were detected using anti-FLAG-M2 monoclonal antibody (dilution 1:500) (Sigma); Substrate protein levels were detected using polyclonal anti-MLC PLoS ONE | www.plosone.org 9 February 2011 | Volume 6 | Issue 2 | e17344