DRP-1b - A Novel ZIPk-Like Isoform Figure 8. A model of the Evolution of the DAP kinases. Scheme showing a most-parsimonious model of the evolution of DRP-1 and ZIPk in vertebrates. doi:10.1371/journal.pone.0017344.g008 antibody (dilution 1:300) (E201); MLC-phosphorylation was detected using polyclonal anti-phospho Ser19 MLC antibody (dilution 1:1,000) (Cell Signaling). Immunostaining 0.86106 HeLa cells were seeded on glass cover slips in 9 cm plates and transfected the next day with the appropriate constructs, 10 mg DNA per plate. After 24 hours, cells were fixed in 3.7% formaldehyde for 15 min. After blocking and permeabi- lization with 10% normal goat serum (Biological Industries), 0.4% Triton X-100 in PBS, the cells were incubated for 1 h with anti- FLAG polyclonal antibody (Sigma; 1:600 dilution) followed by RRX-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch; dilution 1:800). The cover slips were finally stained with DAPI (0.5 mg/ml, Sigma) and mounted with ImmuMount (Thermo Shandon) embedding media. Stained cells were viewed by fluorescent microscopy (Olympus BX41) equipped with a 100x oil immersion objective, using excitation wavelengths of 530-550l (for RRX) and 360-370l (for DAPI). Digital imaging was performed with a DP50 CCD camera using Viewfinder Lite and Studio Lite software (Olympus). Final composites were prepared in Adobe Photoshop (Adobe Systems). Transmission Electron Microscope 293T cells were transfected with DRP-1b, DRP-1 or ZIPk expression plasmids. Cells were fixed for 1 hr in Karnovsky’s fixative (3% paraformaldehyde, 2% glutaraldehyde, 5 mM CaCl2 in 0.1 M cacodylate buffer [pH 7.4], containing 0.1 M sucrose). Cells were scraped, pelleted, and embedded with agar noble to a final concentration of 1.7% and postfixed with 1% OsO4, 0.5% potassium dichromate, and 0.5% potassium hexacyanoferrate in 0.1 M cacodylate buffer. The pellet was stained en bloc with 2% aqueous uranyl acetate followed by ethanol dehydration and embedded in EMbed (EMS). Sections (75 nm) were cut, stained with 2% uranyl acetate in 50% ethanol and lead citrate, and examined using a T12 BioTwin (FEI Holand) transmission electron microscope at an accelerating voltage of 120 KV. Digital images were obtained with Eagle CCD 2K by 2K camera (FEI Holand). Supporting Information Figure S1 DRP-1b proteins alternatively-spliced extra catalytic region. Most fish species included have two DRP-1b isoforms marked by 1 and 2. See text for discussion of the fish DRP-1b isoforms. (DOCX) Figure S2 Conservation of the leucine zipper-like motif in DRP-1b and ZIPk. Logo showing the conservation of the leucine zipper-like motif of both proteins. Upper case letters colors indicate amino acid sub group; lower case letters indicate the amino acid position in the a-helix structure, with the d position of the hydrophobic amino acid marked in red. Sequence logos were calculated according to reference [Henikoff, S., Henikoff, J. G., Alford, W. J., and Pietrokovski, S. (1995) Gene (Amst.) 163, GC17–GC26]. (TIF) PLoS ONE | www.plosone.org 10 February 2011 | Volume 6 | Issue 2 | e17344